Nucleic acid sequences encoding human slit-, megf-, and roundabout-like polypeptides

ABSTRACT

This application is drawn to novel nucleic acid sequences encoding mammalian polypeptides that have sequence similarity to slit-, MEGF- or multiple epidermal growth factor, and roundabout-proteins. The nucleic acid sequence is 2341 nucleotides long and contains an open reading frame from nucleotides 215 to 2173. The encoded polypeptides of 653 residues are novel.

RELATED APPLICATIONS

This application is a continuation and claims priority to U.S. Ser. No.09/520,781, filed Mar. 8, 2000, now U.S. Pat. No. 6,689,866, issued Feb.10, 2004, and PCT/US00/06280, filed Mar. 9, 2000, published, both ofwhich claim the benefit of U.S. Ser. No. 60/123,667, filed Mar. 9, 1999.

FIELD OF THE INVENTION

The invention relates to polynucleotides and secreted ormembrane-associated polypeptides encoded by such polynucleotides, aswell as vectors, host cells, antibodies and recombinant methods forproducing the polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

Eukaryotic cells are subdivided by membranes into multiple functionallydistinct compartments that are referred to as organelles. Each organelleincludes proteins essential for its proper function. These proteins caninclude sequence motifs often referred to as sorting signals. Thesorting signals can aid in targeting the proteins to their appropriatecellular organelle(s). In addition, sorting signals can direct someproteins to be exported, or secreted, from the cell.

One type of sorting sequence is a signal sequence (also referred to as asignal peptide or leader sequence). The signal sequence is present as anamino terminal extension on a newly synthesized polypeptide chain. Asignal sequence targets proteins to an intracellular organelle calledthe endoplasmic reticulum (ER).

The signal peptide takes part in an array of protein-protein andprotein-lipid interactions that result in translocation of a polypeptidecontaining the signal sequence through a channel in the ER. Aftertranslocation, a membrane-bound enzyme (signal peptidase) liberates themature protein from the signal sequence.

The ER functions to separate membrane-bound proteins and secretedproteins from proteins that remain in the cytoplasm. Once targeted tothe ER, both secreted and membrane-bound proteins can be furtherdistributed to another cellular organelle called the Golgi apparatus.The Golgi directs the proteins to vesicles, lysosomes, the plasmamembrane, mitochondria and other cellular organelles.

Only a limited number of genes encoding human membrane-bound andsecreted proteins have been identified. Examples of known secretedproteins include human insulin, interferon, interleukins, transforminggrowth factor-beta, human growth hormone, erythropoietin, lymphokines. Aneed exists for identifying and characterizing additional novel humansecreted proteins and the genes that encode them.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of novelhuman polynucleotide sequences and the membrane-bound or secretedpolypeptides encoded by these sequences. Polypeptides of the inventioninclude a chemokine receptor-like protein (clone 2777610), semaphorinprotein-like splice variants (assembled clones 2864933-1 and 2864933-2,and the pCEP4/Sec-2864933 vector and cDNA clone pCR2.1-2864933), aputative mitochondrial protein (clone 2982339), SLIT protein-like splicevariants (assembled clones 3352358-1 and 3352358-2 and the cDNA clone3352358-S153A), a putative microbody (peroxisome) associated protein(clones 3884846, 3884846-1 and 3884846-2), a tetraspanin-like protein(clones 3911675 and 3911675-2), a putative proline-rich membrane protein(clones 4004056 and 4004056.0.143u), a laminin β-chain precursor-likeprotein (clone 4004731-1), AVENA protein-like splice variants (clones4009334-1 and 4009334-2), a fetal lung-associated protein (clone4035508) and a myeloid upregulated protein (clone 4339264). Thesepolynucleotides and the polypeptides encoded thereby are collectivelyreferred to as the SECX gene set, the sequences of which are disclosedin SEQ ID NOs:1-32.

In one aspect, the invention includes an isolated SECX nucleic acidmolecule which includes a nucleotide sequence encoding a polypeptidethat includes the amino acid sequence of one or more of SEQ ID NOs:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 75, 77, 79 and 81.For example, in various embodiments, the nucleic acid can include anucleotide sequence that includes SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 74, 76, 78 and 80. Alternatively, theencoded SECX polypeptide may have a variant amino acid sequence, e.g.,have an identity or similarity less than 100% to the disclosed aminoacid sequences, as described herein.

The invention also includes an isolated polypeptide that includes theamino acid sequence of one or more of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14,16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48: or afragment having at least 15 amino acids of these amino acid sequences.Also included is a naturally occurring polypeptide variant of a SECXpolypeptide, wherein the polypeptide is encoded by a nucleic acidmolecule which hybridizes under stringent conditions to a nucleic acidmolecule consisting of a SECX nucleic acid molecule.

Also included in the invention is an antibody which selectively binds toa SECX polypeptide.

The invention further includes a method for producing a SECX polypeptideby culturing a host cell expressing one of the herein described SECXnucleic acids under conditions in which the nucleic acid molecule isexpressed.

The invention also includes methods for detecting the presence of a SECXpolypeptide or nucleic acid in a sample from a mammal, e.g., a human, bycontacting a sample from the mammal with an antibody which selectivelybinds to one of the herein described polypeptides, and detecting theformation of reaction complexes including the antibody and thepolypeptide in the sample. Detecting the formation of complexes in thesample indicates the presence of the polypeptide in the sample.

The invention further includes a method for detecting or diagnosing thepresence of a disease, e.g., a pathological condition, associated withaltered levels of a polypeptide having an amino acid sequence at least80% identical to a SECX polypeptide in a sample. The method includesmeasuring the level of the polypeptide in a biological sample from themammalian subject, e.g., a human, and comparing the level detected to alevel of the polypeptide present in normal subjects, or in the samesubject at a different time, e.g., prior to onset of a condition. Anincrease or decrease in the level of the polypeptide as compared tonormal levels indicates a disease condition.

Also included in the invention is a method of detecting the presence ofa SECX nucleic acid molecule in a sample from a mammal, e.g., a human.The method includes contacting the sample with a nucleic acid probe orprimer which selectively hybridizes to the nucleic acid molecule anddetermining whether the nucleic acid probe or primer binds to a nucleicacid molecule in the sample. Binding of the nucleic acid probe or primerindicates the nucleic acid molecule is present in the sample.

The invention further includes a method for detecting or diagnosing thepresence of a disease associated with altered levels of a SECX nucleicacid in a sample from a mammal, e.g,. a human. The method includesmeasuring the level of the nucleic acid in a biological sample from themammalian subject and comparing the level detected to a level of thenucleic acid present in normal subjects, or in the same subject at adifferent time. An increase or decrease in the level of the nucleic acidas compared to normal levels indicates a disease condition.

The invention also includes a method of treating a pathological state ina mammal, e.g,. a human, by administering to the subject a SECXpolypeptide to the subject in an amount sufficient to alleviate thepathological condition. The polypeptide has an amino acid sequence atleast 80% identical to a SECX polypeptide.

Alternatively, the mammal may be treated by administering an antibody asherein described in an amount sufficient to alleviate the pathologicalcondition.

Pathological states for which the methods of treatment of the inventionare envisioned include a cancer, e.g., colorectal carcinoma, a prostatecancer a benign tumor, an immune disorder, an immune deficiency, anautoimmune disease, acquired immune deficiency syndrome, transplantrejection, allergy, an infection by a pathological organism or agent, aninflammatory disorder, arthritis, a hematopoietic disorder, a skindisorder, atherosclerosis, restenosis, a neurological disease,Alzheimer's disease, trauma, a surgical or traumatic wound, a spinalcord injury, and a skeletal disorder.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the nucleotide and encoded polypeptidesequences of clone 2777610.

FIG. 2 is a representation of the nucleotide and encoded polypeptidesequences of clone 2864933-1.

FIG. 3 is a representation of the nucleotide and encoded polypeptidesequences of clone 2864933-2.

FIG. 4 is a representation of the nucleotide and encoded polypeptidesequences of clone 2982339.

FIG. 5 is a representation of the nucleotide and encoded polypeptidesequences of clone 3352358-1.

FIG. 6 is a representation of the nucleotide and encoded polypeptidesequences of clone 3352358-2.

FIG. 7 is a representation of the nucleotide and encoded polypeptidesequences of clone 3884846 (FIG. 7A), clone 3884846-1 (FIG. 7B), andclone 3884846-2 (FIG. 7C).

FIG. 8 is a representation of the nucleotide and encoded polypeptidesequences of clone 3911675 (FIG. 8A) and clone 3911675-2 (FIG. 8B).

FIG. 9 is a representation of the nucleotide and encoded polypeptidesequences of clone 4004056 (FIG. 9A) and clone 4004056.0.143u (FIG. 9B).

FIG. 10 is a representation of the nucleotide and encoded polypeptidesequences of clone 4004731-1.

FIG. 11 is a representation of the nucleotide and encoded polypeptidesequences of clone 4009334-1.

FIG. 12 is a representation of the nucleotide and encoded polypeptidesequences of clone 4009334-2.

FIG. 13 is a representation of the nucleotide and encoded polypeptidesequences of clone 4035508.

FIG. 14 is a representation of the nucleotide and encoded polypeptidesequences of clone 4339264.

FIG. 15 is a representation of the nucleotide and encoded polypeptidesequences of the cDNA clone pCR2.1-2864933.

FIG. 16 depicts the Western blot after reducing SDS-PAGE of expressionof the pCEP4/Sec-2864933 vector in 293 cells.

FIG. 17 depicts the nucleotide (panel A) and amino acid (panel B)sequences obtained for the cDNA clone 3352358-S153A, which comprises theextracellular domain of 3352358-1, wherein the underlined sequencesdepict flanking sequence.

FIG. 18 depicts expression of the pCEP4/Sec-3352358 vector in 293 cellsanalyzed in a Western blot of 293 cell extracts after reducing SDS-PAGE.

FIG. 19 depicts real time quantitative PCR (TaqMan™) analysis of theexpression of clone 2864933 utilizing primer-probe set 88 (Panel A),primer-probe set 291 (Panel B), and primer-probe set 341 (Panel C).

FIG. 20 depicts real time quantitative PCR (TaqMan™) analysis of theexpression of 3352358.

FIG. 21 depicts real time quantitative PCR (TaqMan™) analysis of theexpression of 3911675.

FIG. 22 depicts real time quantitative PCR (TaqMan™) analysis of theexpression of 4035508.

FIG. 23 depicts real time quantitative PCR (TaqMan™) analysis of theexpression of 4339264.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, upon the discovery of novelpolynucleotide sequences and the membrane-bound or secreted polypeptidesencoded by these sequences. Polypeptides encoded by nucleotides of theinvention include clone 2777610 (a chemokine receptor-like protein, SEQID NOS:1-2); assembled clones 2864933-1 and 2864933-2 and thepCEP4/Sec-2864933 vector and cDNA clone pCR2.1-2864933 (semaphorinprotein-like splice variants, SEQ ID NOS:3-6, 29-30); clone 2982339 (aputative mitochondrial protein, SEQ ID NOS:7-8), assembled clones3352358-1 and 3352358-2 and the cDNA clone 3352358-S 153A (SLITprotein-like splice variants, SEQ ID NOS:9-12, 31-32); clones 3884846,3884846-1 and 3884846-2 (putative microbody/peroxisome associatedprotein splice variants, SEQ ID NOS:13-14 and 74-77), clones 3911675 and3911675-2 (tetraspanin-like protein splice variants, SEQ ID NOS:15-16and 78-79); clones 4004056 and 4004056.0.143u (putative proline-richmembrane protein splice variants, SEQ ID NOS:17-18 and 80-81); clone4004731-1 (a laminin β-chain precursor-like protein, SEQ ID NOS:19-20);clones 4009334-1 and 4009334-2 (AVENA protein-like splice variants, SEQID NOS:21-24); clone 4035508 (a novel fetal lung-associated protein, SEQID NOS:25-26); and clone 4339264 (a myeloid upregulated protein, SEQ IDNOS:27-28). These genes are collectively referred to as the SECX geneset. The polynucleotides and polypeptides are set forth in Table 1.Table 1 lists the SEQ ID NOs for each nucleotide and amino acid sequenceof the invention, as well as SEQ ID NOs for the primers specific to theclones of this invention that were employed in various aspects andembodiments described herein.

TABLE 1 Sequences and Corresponding SEQ ID Numbers Nucleic acid ProteinClone or Primer No. Figure SEQ ID NO: SEQ ID NO: 2777610  1 1 22864933-1  2 3 4 2864933-2  3 5 6 2982339  4 7 8 3352358-1  5 9 103352358-2  6 11 12 3884846 7A 13 14 3884846-1 7B 74 75 3884846-2 7C 7677 3911675 8A 15 16 3911675-2 8B 78 79 4004056 9A 17 18 4004056.0.143u9B 80 81 4004731-1 10 19 20 4009334-1 11 21 22 4009334-2 12 23 244035508 13 25 26 4339264 14 27 28 pCR2.1-2864933 15 29 30 3352358-S153A17A, 17B 31 32 2864933 MatF 33 2864933 F-TOPO-Reverse 34 2864933-Seq-035 2864933-Seq-1 36 2864933-Seq-2 37 2864933-Seq-3 38 2864933-Seq-4 392864933-Seq-5 40 2864933-Seq-6 41 pSec-V5-His Forward 42 pSec-V5-HisReverse 43 3352358CForward 44 3352358CReverse 45 3352358 Seq-1 463352358 Seq-2 47 3352358 Seq-3 48 3352358 Seq-4 49 Ag 111 (F) 50 Ag 111(R) 51 Ag 111 (P) 52 Ag 88 (F) 53 Ag 88 (R) 54 Ag 88 (P) 55 Ag 291 (F)56 Ag 291 (R) 57 Ag 291 (P) 58 Ag 341 (F) 59 Ag 341 (R) 60 Ag 341 (P) 61Ag 42 (F) 62 Ag 42 (R) 63 Ag 42 (P) 64 Ag 115 (F) 65 Ag 115 (R) 66 Ag115 (P) 67 Ag 118 (F) 68 Ag 118 (R) 69 Ag 118 (P) 70 Ag 120 (F) 71 Ag120 (R) 72 Ag 120 (P) 731. Clone 2777610

Clone 2777610 is a 1812 bp nucleic acid sequence (SEQ ID NO:1) that wasoriginally identified in bone tissue, which includes bone marrow. Thefull length clone (FIG. 1) was further assembled from sequencesexpressed in bone tissues. An open reading frame (“ORF”) encoding apolypeptide (SEQ ID NO:2) having 333 amino acid residues is found atnucleotides 537-1535 (FIG. 1). The nucleotide sequence includes a Kozaksequence. The stop codon TGA is found at nucleotides 1536-1538. Theresults of a PSORT analysis predict that the protein is localized in theplasma membrane with a certainty of 0.6000. The SignalP program predictsthat a signal sequence occurs with a most likely cleavage site betweenresidues 44 and 45, represented by the dash between the amino acidsTLA-LW (i.e., ThrLeuAla-LeuTrp).

The protein of clone 2777610 has 332 of 333 residues both (99%)identical and positive to a human seven transmembrane receptor proteindesignated HNEAA81 (European Patent number 913471-A2). As used herein,“identical” residues correspond to those residues in a comparisonbetween two sequences where the equivalent nucleotide base or amino acidresidue in an alignment of two sequences is the same residue. Residuesare “positive” when the comparisons between two sequences in analignment show that residues in an equivalent position in a comparisonare either the same amino acid or a conserved amino acid as definedbelow. Clone 2777610 was also found to have 328 of 333 residues both(98%) identical and positive to a human chemokine receptor-like protein(PCT Publication WO9839441-A1). A weaker similarity was also detectedfor a 338 residue human probable G protein-coupled receptor KIAA0001(GenBank Accession number Q15391).

Members of the G protein-coupled receptor (GPCR) superfamily containseven transmembrane domains and transduce extracellular signals throughheterotrimeric G proteins. G-protein-coupled receptors (GPCRs) areintegral membrane proteins of great pharmacological importance owing totheir central role in the regulation of cellular responses to externalstimuli. See, for example, Marchese et al., 1999 Trends Pharmacol Sci20(9): 370-5; and Rozengurt 1998 J Cell Physiol 177(4):507-17.

GPCR receptors specifically bind select neurotransmitters and peptidehormones, and are likely to underlie the recognition andG-protein-mediated transduction of various signals. These signalsactivated by ligand-bound GPCRs have been implicated in a variety ofnormal and abnormal processes, including development, inflammation, andmalignant transformation (matrix invasion, motility, chemotaxis,adhesion, growth and survival signaling). These signaling peptides exerttheir characteristic effects on cellular processes by binding tospecific GPCRs on the surface of their target cells. Typically, thebinding of a neuropeptide to its cognate GPCR triggers the activation ofmultiple signal transduction pathways that act in a synergistic andcombinatorial fashion to relay the mitogenic signal to the nucleus andpromote cell proliferation. A rapid increase in the synthesis oflipid-derived second messengers with subsequent activation of proteinphosphorylation cascades is an important early response toneuropeptides. An emerging theme in signal transduction is that theseagonists also induce rapid and coordinate tyrosine phosphorylation ofcellular proteins including the nonreceptor tyrosine kinase p125fak andthe adaptor proteins p130cas and paxillin. This tyrosine phosphorylationpathway depends on the integrity of the actin cytoskeleton and requiresfunctional Rho.

The Clone 2777610 protein is a seven transmembrane receptor protein withchemokine receptor-like properties. Clone 2777610 is a G protein coupledreceptor (GPCR) that is a likely gamma-aminobutyric acid receptor, inthe class of P2Y-like GPCRs. As such, Clone 2777610 is useful indiagnosing and/or treating pathologies and disorders associated withG-protein coupled receptor metabolism, e.g., bacterial disease; asthma;fungal disease; viral disease; HIV-1; HIV-2; cancer; anorexia;Parkinson's disease; hypertension; osteoporosis; myocardial infarction;manic depression; schizophrenia; Gilles dela Tourett's syndrome;inflammatory disorder; and viral infection.

Based on the roles of other GPCRs and the high expression of 2777610 inlymphoid tissue such as the spleen, bone marrow, lymph node (see Example6), the inventor anticipates that successful therapeutic targeting of2777610, using either small molecules that inhibit transmembranesignaling by 2777610 or monoclonal antibodies designed to block theinteraction of 2777610 with ligand(s), might have utility in modulatinglymphoproliferative disorders (myeloma, myeloid leukemia, non-Hodgkin'slymphoma, etc.) or autoimmune diseases (SLE, etc.) or both. Likewise,with respect to the high expression of 2777610 in whole adult humanbrain, hippocampus, substantia nigra and spinal cord, modulation of2777610 signaling using the approaches described above will haveclinical utility to modulate certain neurodegenerative disordersaffecting motor function.

2. Clone 2864933-1

Clone 2864933-1 includes a nucleic acid sequence (SEQ ID NO:3) including3498 nucleotides (FIG. 2). This clone is similar to clone 2864933-2(below) with the exception that 2864933-1 has an insert of 164nucleotides at positions 1942-2106. The gene fragment giving rise tothis clone was found in mainly in heart tissue. Fragments included inthis gene were also found in lymph node, pancreas, thalamus, brain,salivary gland and adrenal gland. Clone 2864933-1 includes a Kozaksequence, a start codon at nucleotides 214-216, and a TAA terminationcodon at nucleotides 3031-3033. The nucleotide residues between 214-3030define an ORF encoding a protein (SEQ ID NO:4) of 939 amino acidresidues (FIG. 2). Molecular cloning and expression of a fragmentcorresponding to the putative mature extracellular domain of 2864933-1is given in Examples 2 and 3. The PSORT program predicts that the2864933-1 protein localizes to the plasma membrane with a certainty of0.4600. The protein is a likely Type I transmembrane protein, with thepredicted transmembrane domain between residues 645-661 of SEQ ID NO:4.The SignalP program predicts that the protein has a signal peptidecleavage site between residues 18 and 19, represented by the dashbetween the amino acids AGA-GF (i.e., AlaGlyAla-Gly Phe).

The 2864933-1 protein is 94% identical, and 97% similar, to a murinesemaphorin polypeptide having 888 amino acid residues (GenBank AccessionNumber AAB86408). In addition, it shows 35% identity and 53% similarityto human semaphorin III (GenBank Accession Number AAA65938). For thesereasons the 2864933-1 polypeptide is believed to be a cytokine-likegrowth factor.

The semaphorin (a.k.a. collapsin) family of molecules plays a criticalrole in the guidance of nerve growth cones during neuronal development.The family is characterized by the presence of a conserved semaphorindomain at the amino terminus. Mutational analysis of human semaphorinA(V) revealed mutations (germline in 1 case) in 3 of 40 lung cancers.Semaphorin E is responsible for a non-MDR drug resistance in humancancers including ovarian cancer and is overexpressed in CDDP-resistantcell lines as well as induced by diverse chemotherapeutic drugs and byX-ray and UV irradiation. Yamada et al. 1997 Proc. Nat. Acad. Sci. 94:14713-14718. Human semaphorin E mRNA is up-regulated in synovialfibroblasts of rheumatoid arthritis patients. Mangasser-Stephan et al.1997 Biochem. Biophys. Res. Commun. 234: 153-156. Human neuropilin-1, areceptor for the collapsin/semaphorin family that mediates neuronal cellguidance, is expressed by endothelial and tumor cells as anisoform-specific receptor for vascular endothelial growth factor and isbelieved to regulate VEGF-induced angiogenesis. Soker et al. 1998 Cell92: 735-745.

Semaphorins, the plexin family of semaphorin receptors, and scatterfactor receptors share evolutionarily conserved protein modules, e.g.the semaphorin domain and Met Related Sequences (MRS). Artigiani et al.,1999, IUBMB Life 48(5):477-82. These proteins have a common role ofmediating cell guidance cues. During development, scatter factorreceptors control cell migration, epithelial tubulogenesis, and neuriteextension. Semaphorins and their receptors are known signals for axonguidance. They are also believed to regulate developmental processesinvolving cell migration and morphogenesis, and have been implicated inimmune function and tumor progression. Scatter factors and secretedsemaphorins are diffusible ligands, whereas membrane-bound semaphorinssignal by cell-cell interaction. Cell guidance control by semaphorinsrequires plexins, alone or in a receptor complex with neurophilins.Semaphorins, besides their role in axon guidance, are expected to havemultiple functions in morphogenesis and tissue remodeling by mediatingcell-repelling cues through plexin receptors.

The potential roles of the 2864933 protein in tumorgenesis includedevelopment of chemoresistance, radiotherapy resistance, survival introphic factor limited secondary tissue site microenvironments,potential involvement in enhancing VEGF-induced angiogenesis.

Based on the reported roles of semaphorins summarized herein it isanticipated that successful therapeutic targeting of 2864933 and/or itssplice variants will result in significant anti-tumor activity incombination with established cytotoxic/genotoxic therapies (i.e.chemosensitization, radiosensitization). Additionally, the semaphorinsplay roles in axon outgrowth and neuronal cell migration. In this regardsuccessful therapeutic targeting of 2864933-1 and/or 2864933-2 mightalso limit the extent (frequency) of metastatic dissemination (tumorburden) and potentially limit tumor angiogenesis. Therapeutic targetingof 2864933 and its splice variants is also provided via the generationof human or humanized monoclonal antibodies that block the ability of2864933-1 or 2864933-2 to interact with cognate ligand(s) and elicit atransmembrane signal(s). Equally, the generation of small molecules(synthetics, cell permeable peptides, other) than specifically interferewith one or more of the downstream signaling components in thepathway(s) activated by ligand-bound 2864933-1 and/or 2864933-2 would beexpected to have significant anti-tumor activity as described above.Likewise, the introduction of antisense constructs (naked DNA,adenoviral constructs), ribozymes to inhibit the expression of 2864933-1and/or 2864933-1 would be expected to have significant anti-tumoractivity as described above.

Based on the expression profiles of the 2864933-1 and 2864933-2transcripts set forth in Example 7 and FIGS. 19A, 19B and 19C,therapeutic indications for targeting 2864933-1 and 2864933-2 includerenal cell carcinomas, small cell lung cancers, large cell variants ofsmall cell lung cancer, breast adenocarcinomas, and malignant melanomas.Clones 2864933-1 and 2864933-2 are useful in diagnosing and/or treatingpathologies related to developmental malfunction, especially in thenervous system, and in treatment of CNS pathologies, e.g., Alzheimer'sdisease and parkinsonism.

3. Clone 2864933-2

Clone 2864933-2 has a nucleic acid sequence (SEQ ID NO:5) of 3333nucleotides (FIG. 3). This clone is similar to clone 2864933-1 (above;FIG. 2) with the exception that 2864933-1 has an insert of 164nucleotides at positions 1942-2106(according to the SEQ ID NO:3numbering for clone 2864933-1). This difference appears to be an RNAsplicing variation. The gene fragment giving rise to this clone2864933-2 was found mainly in heart tissue. Transcribed sequences fromthis gene are also found in lymph node, pancreas, thalamus, brain,salivary gland and adrenal gland. Clone 2864933-1 includes a Kozaksequence, a start codon at nucleotides 214-216, and a TAA terminationcodon at nucleotides 2866-2868. The nucleotides between 214 and 2865thus define an ORF encoding a protein (SEQ ID NO:6) of 884 amino acidresidues (FIG. 3). The PSORT program predicts that the 2864933-1 proteinlocalizes to the plasma membrane with a certainty of 0.4600. The SignalPprogram predicts that the protein most likely has a signal peptidecleavage site between residues 18 and 19, represented by the dashbetween the amino acids AGA-GF (i.e., AlaGlyAla-GlyPhe).

The 2864933-2 protein is 95% identical, and 97% similar to murinesemaphorin having 888 amino acid residues (GenBank Accession numberAAB86408). In addition, it shows 38% identity and 55% similarity tohuman semaphorin III (GenBank Accession number AAA65938).

The 2864933-2 protein was also found to have 869 of 877 residues (99%)identical, and 871 of 877 residues (99%) positive, to the 974 residuehuman secreted protein from a clone designated CJ145-1 (PCT PublicationWO9827205-A2). The 2864933-2 sequence was isolated from a human fetalbrain cDNA library and is a novel secreted protein distinct from cloneCJ145-1 in both size and sequence. Clone 2864933-2 is useful forcytokine and cell proliferation/differentiation activity, immunestimulating or suppressing activity, hematopoiesis regulating activity,tissue growth activity, activin/inhibin activity,chemotactic/chemokinetic activity, hemostatic and thrombotic activity,receptor/ligand activity, anti-inflammatory activity, cadherin/tumorinvasion suppressor activity, tumor inhibition activity and otheractivities. The Clone 2864933-2 is also useful in diagnosing and/ortreating pathologies related to developmental malfunction, especially inthe nervous system, and in the treatment of CNS pathologies, e.g.,Alzheimer's disease and parkinsonism.

4. Clone 2982339

Clone 2982339 has a sequence (SEQ ID NO:7) of 856 nucleotides (FIG. 4),including a Kozak sequence, an initiation codon at positions 138-140 anda TGA stop codon at positions 726-728. This sequence between residues138 to 725 defines an open reading frame encoding a protein (SEQ IDNO:8) of 196 amino acid residues (FIG. 4). The clone originated fromfetal brain and was assembled using 65 sequences from fetal thymus andplacenta. Fragments for this clone are also found in human placenta,thymus gland, thyroid gland, and bone, including osteosarcomas. ThePSORT predicts that the 2982339 protein localizes to the mitochondrialmatrix space with a certainty of 0.7077. SignalP suggests that theprotein may have no known N-terminal signal sequence.

The 2982339 protein has 16 of 54 residues (29%) identical to, and 24 of54 residues (44%) positive with, an artificial sequence of 109 residuesthat is an aprotinin analogue precursor (GenBank Accession numbersAAB54954 and AAB54956). Aprotinin, also known as pancreatic trypsininhibitor precursor or basic protease inhibitor, is an intracellularpolypeptide found in many tissues, and is a known inhibitor of trypsin,kallikrein, chymotrypsin, and plasmin. GenBank Accession number P00974;Creighton and Charles, 1987 J. Mol. Biol. 194 (1): 11-22.

5. Clone 3352358-1

Clone 3352358-1 includes a 2341 nucleotide sequence (SEQ ID NO:9) (FIG.5) with an initiation codo at nucleotides 215-217 and a TAA stop codonat nucleotides 2174-2176. This sequence between nucleotides 215 to 2173defines an ORF encoding a protein (SEQ ID NO:10) of 653 residues (FIG.5). The clone was identified by a polynucleotide fragment originating infetal liver. Expressed sequences are also found in liver, includingfetal liver, kidney, including fetal kidney, and thalamus. The PSORTprogram predicts that the 3352358-1 protein localize in the plasmamembrane with a certainty of 0.46. The SignalP program predicts that theprotein as a signal peptide, with the most likely cleavage site betweennucleotides 38 and 39, represented by the dash between the amino acidsAAA-AS (i.e., AlaAlaAla-AlaSer), or between nucleotides 41 and 42,represented by the dash between the amino acids ASA-GP (i.e.,AlaSerAla-GlyPro). The protein is predicted to be a Type I transmembraneprotein with the transmembrane domain located between nucleotides 522and 551.

The 3352358-1 protein has 35% of its residues identical, and 48% of itsresidues similar to, human slit-1 protein, a protein of 1534 residues(GenBank Accession number BAA35184). 3352358-1 protein is also 39%identical and 46% similar to human slit-3 protein, a protein of 1523residues (GenBank Accession number BAA35186); and 40% identical and 48%similar to the human neurogenic extracellular slit protein slit-2 having1521 residues (GenBank Accession number AAD04309). The 3352358-1 proteinhas an overall 53% identity to a hypothetical 45.1 kDa protein (GenBankAccession number CAB70473).

The slit genes encode proteins with a conserved chemorepulsive activityfor axons in invertebrates and vertebrates. Chen et al., 2000,Neuroscience 96: 231-236; Yuan et al., 1999 Dev Biol 212: 290-306. Forexample, the binding of Slit to Roundabout, expressed on the cellsurface, is implicated in neuronal guidance activity. Thus, Slitproteins may guide axon projections in multiple regions of the embryo.

By analogy, Clone 3352358-1 has diagnostic and therapeutic utility inpathologies related to neural development and in CNS pathologies, e.g.,Alzheimer's disease and parkinsonism.

Molecular cloning and expression of the putative mature extracellulardomain of 3352358-1 is described in Examples 4 and 5. This clonedfragment originated from cDNA samples obtained from human testis andfetal brain. The resulting clone, designated clone 3352358-S153A,differs in sequence from that shown in FIG. 5. The respective3352358-S153A nucleotide sequence is disclosed in FIG. 17A (SEQ IDNO:31) and polypeptide sequence is disclosed in FIG. 17B (SEQ ID NO:32). One reason for the sequence difference between the 3352358-1 cloneand 3352358-S153A cDNA is likely the tissue or organ sources of thecDNAs. If so, this finding represents a tissue-specific ororgan-specific basis for allelic variants (also known as isoforms) ofproteins, e.g., the disclosed 3352358-1 slit-like protein. The 3352358-1and 3352358-S153A clones will thus have utility in identifying thosetissue or cell types that express these allelic or splice variants.

The 3352358 sequence is related to MEGF (multiple epidermal growthfactor-like domains)/Slit family and roundabout. The domain thatcharacterizes epidermal growth factor consists of approximately 50 aminoacids with 3 disulfide bonds. EGF-like domains are believed to play acritical role in a number of extracellular events, including celladhesion and receptor-ligand interactions. Proteins with EGF-likedomains often consist of more than 1,000 amino acids, have multiplecopies of the EGF-like domain, and contain additional domains known tobe involved in specific protein-protein interactions.

Important members of this family include fat tumor suppressor,(Drosophila, homolog of, 2; fat2). TheDrosophila fat gene is a tumorsuppressor gene whose product controls cell proliferation andmorphogenesis in the imaginal discs in a contact-dependent manner.Another relative of 3352358 is Slit1 (also known as MEGF4), a Drosophilagene involved in the formation and maintenance of the nervous andendocrine systems. Another relative of 3352358 is roundabout, aDrosophila gene that controls axon crossing of the CNS midline anddefines a novel subfamily of evolutionarily conserved guidancereceptors. Kidd et al. 1998 Cell 92: 205-215; Nakayama et al. 1998Genomics 51: 27-34.

The potential role(s) of 3352358 in Tumorgenesis includechemoresistance, radiotherapy resistance, survival in trophic factorlimited secondary tissue site microenvironments, potential involvementin angiogenesis.

Based on the reported roles of MEGFs/SLITs/Roundabout described hereinit is anticipated that successful therapeutic targeting of 3352358and/or its splice variants will result in significant anti-tumoractivity in combination with established cytotoxic/genotoxic therapies(i.e. chemosensitization, radiosensitization). Additionally, thesemaphorins play roles in axon outgrowth and neuronal cell migration. Inthis regard successful therapeutic targeting of 3352358 might also limitthe extent (frequency) of metastatic dissemination (tumor burden) andpotentially limit tumor angiogenesis. Therapeutic targeting of 3352358and its splice variants will also be provided via the generation ofhuman or humanized monoclonal antibodies that block the ability of3352358 to interact with cognate ligand(s) and elicit a transmembranesignal(s). Equally, the generation of small molecules (synthetics, cellpermeable peptides, other) than specifically interfere with one or moreof the downstream signaling components in the pathway(s) activated byligand-bound 3352358 will have significant anti-tumor activity asdescribed above. Likewise, the introduction of antisense constructs(naked DNA, adenoviral constructs), ribozymes to inhibit the expressionof 3352358 will have significant anti-tumor activity as described above.

Based on the expression profiles of the 3352358 transcripts presented inExample 8 and FIG. 20, the therapeutic indications for targeting 3352358include select hepatomas/hepatocellular carcinomas and renal cellcarcinomas.

6. Clone 3352358-2

Clone 3352358-2 of 2607 nucleotides (SEQ ID NO:11) includes a Kozaksequence, an initiation codon at nucleotides 215-217 and a TAAtermination codon at nucleotides 1985-1987 (FIG. 6). This sequencebetween residues 215 to 1984 defines an ORF encoding a protein (SEQ IDNO:12) of 590 residues (FIG. 6). The PSORT program predicts that the3352358-2 localizes in the plasma membrane. The SignalP program predictsthat the protein has a signal peptide, with the most likely cleavagesite between residues 38 and 39, represented by the dash between theamino acids AAA-AS (i.e., AlaAlaAla-AlaSer). This clone originates inhuman liver, including adult and fetal liver. Transcribed sequences fromthis gene are found in liver, including fetal liver, bone, includingbone marrow, brain, and the pituitary gland.

Similarity searches indicate that the 3352358-2 protein is 35% identicaland 48% similar with human slit-1 protein (GenBank Accession numberBAA35184) having 1534 residues. The slit genes encode proteins with aconserved chemorepulsive activity that affects axons from bothinvertebrates and vertebrates. Chen et al., 2000, Neuroscience96(1):231-236; Yuan et al., 1999 Dev Biol 212(2):290-306. Binding ofSlit to the Roundabout protein expressed on a cell surface is implicatedin this neuronal guidance activity. Thus Slit proteins guide axonprojections in multiple regions of the developing embryo.

Clone 3352358-2 will have diagnostic and therapeutic utility inpathologies related to neural development and CNS pathologies, e.g.,Alzheimer's disease and parkinsonism.

7. Clone 3884846

Clone 3884846 includes a polynucleotide (SEQ ID NO:13) of 1340nucleotides (FIG. 7A) having a Kozak sequence, an initiation codon atnucleotides 421-423 and a TAG stop codon at nucleotides 1288-1290. Thissequence between residues 421 through 1287 defines an ORF encoding aprotein (SEQ ID NO:14) of 289 amino acid residues (FIG. 7A). Transcribedsequences from this gene are found in pituitary gland, testis, kidney,including fetal kidney, brain, including fetal brain, pituitary gland,placenta, pancreas, testis, spleen kidney, including fetal kidney, fetalliver, skeletal muscle, heart, OVCAR-3 cells and lung. The PSORT programpredicts that the 3884846 protein localizes to the microbody(peroxisome) with a certainty of 0.7480. There appears to be no knownsignal peptide in the protein.

Clone 3884846-1 does not have an initiation codon at the beginning ofthe nucleotide sequence (SEQ ID NO:74), so it is believed that thedisclosed clone represents an incomplete ORF. It is assumed that aninitiation codon is found upstream from the sequence shown, such thatthe cDNA sequence extends further 5′ from that shown in FIG. 7B. A stopcodon of TGA is located at nucleotides 979-981. The 3884846-1 nucleotidesequence between residues 1 to 978 defines an ORF encoding a protein(SEQ ID NO:75) of 326 amino acid residues (FIG. 7B).

A full length Clone 3884846-2 includes the nucleic acid sequence (SEQ IDNO:76) shown in FIG. 7C. Clone 3884846-2 has an initiation codon atnucleotides 299-301 and a TGA stop codon at nucleotides 983-985. Thissequence between residues 299 to 982 defines an ORF encoding a protein(SEQ ID NO:77) of 228 amino acid residues (FIG. 7C).

8. Clone 3911675

Clone 3911675 is a polynucleotide (SEQ ID NO:15) of 1428 nucleotides(FIG. 8A). The nucleotide sequence includes a Kozak sequence, a startcodon at positions 96-98, and a TGA stop codon at nucleotides 906-908.This sequence between residues 96 through 905 define an ORF encoding aprotein (SEQ ID NO:16) having 270 amino acid residues (FIG. 8A). Theclone originates in DNA isolated from spleen cells. The PSORT programpredicts that the protein is localized in the plasma membrane. Accordingto the SignalP program, the protein is predicted to have a signalpeptide with the most probable cleavage site between 42 and 43,represented by the dash between the amino acids AWS-EK (i.e.,AlaTrpSer-GluLys).

Clone 3911675-2 does not have an initiation codon at the beginning ofthe nucleotide sequence (SEQ ID NO:78), so it is believed that thedisclosed clone represents an incomplete ORF. It is assumed that aninitiation codon is found upstream from the sequence shown, such thatthe cDNA sequence extends further 5′ from that shown in FIG. 8B. A stopcodon of TGA is located at nucleotides 629-631. The 3911675-2 nucleotidesequence between residues 2 to 628 defines an ORF encoding a 3911675-2protein (SEQ ID NO:79) of 209 amino acid residues (FIG. 8B).

In database searches for similarity, the 3911675 protein is 57%identical to, and 75% positive with, human tetraspan NET-4 protein of268 residues (GenBank Accession Number AAC17120), and is 57% identicalto, and 74% positive with, human tetraspanin TSPAN-5 having 264 residues(GenBank Accession Number NP005714).

TM4SF4 (transmembrane 4 superfamily member 4), is an integral membraneglycoprotein found to regulate the adhesive and proliferative status ofintestinal epithelial cells through a density-dependent mechanism.Members of the ‘transmembrane 4 superfamily’ (TM4SF) are cell-surfaceproteins presumed to have 4 transmembrane domains. Many tetraspanproteins are considered “promiscuous” interactors by virtue of theirassociations with other molecules, including lineage-specific proteins,integrins, and other tetraspanins. Tetraspan proteins are involved indiverse processes, e.g., cell activation and proliferation, adhesion andmotility, differentiation, and cancer. Maecker et al. 1997 FASEB J11(6): 428-42. The tetraspan family proteins function as “molecularfacilitators, grouping specific cell-surface proteins and thusincreasing the formation and stability of functional signalingcomplexes” and so aid in the formation of plasma membrane signalingcomplexes. Maecker et al. 1997 FASEB J 11(6): 428-42; Birling et al,1999 J. Neurochem 73(6): 2600-2008. Neuronal tetraspanin family membersare implicated in axon growth and target recognition. Perron and Bixby1999 FEBS Lett 461(1-2): 86-90.

Based on the reported roles of tetraspan-related proteins describedherein it is anticipated that successful therapeutic targeting of3911675 and/or its splice variants will result in significant anti-tumoractivity (tumor growth inhibition) especially in combination withestablished cytotoxic/genotoxic therapies (i.e. chemosensitization,radiosensitization). In this regard successful therapeutic targeting of3911675 might also limit the extent (frequency) of metastaticdissemination (tumor burden) and potentially limit tumor angiogenesis.Therapeutic targeting of 3911675 and its splice variants will also beprovided via the generation of human or humanized monoclonal antibodiesthat block the ability of 3911675 to interact with specific cognateligand(s) and elicit a transmembrane signal(s). Equally, the generationof small molecules (e.g., synthetics, cell permeable peptides) thatspecifically interfere with one or more of the downstream signalingcomponents in the pathway(s) activated by ligand-bound 3911675 will havesignificant anti-tumor activity as described above. Likewise, theintroduction of antisense constructs (naked DNA, adenoviral constructs),ribozymes to inhibit the expression of 3911675 will have significantanti-tumor activity as described above.

Clone 3911675 will thus have diagnostic and therapeutic utility inpathologies related to cell signaling and neural development and in CNSpathologies, e.g., Alzheimer's disease and parkinsonism. Based on theubiquitous expression profiles of the 3911675 gene (see Example 9 andFIG. 21), one specific therapeutic indication for targeting 3911675 isfor malignant melanoma.

9. Clone 4004056

Clone 4004056 includes a nucleic acid sequence (SEQ ID NO:17) of 1767nucleotides (FIG. 9). There is an initiation codon at positions 51-53and a TAA stop codon at positions 984-986. Nucleotides from 51 to 983therefore define an ORF encoding a protein (SEQ ID NO:18) of 311 aminoacid residues (FIG. 9). The clone was originally identified in salivarygland. Transcribed sequences from this gene are found in total RNAlibraries from adrenal gland, placenta, mammary tissue, prostate,testis, uterus, spleen, fetal thymus (CRL7046), osteogenic sarcoma cells(HTB96), fetal lung, thalamus, fetal kidney and Burkitt's lymphoma(i.e., Raji cells), and in mRNA libraries from bone marrow, melanoma,pituitary, thyroid. The PSORT program predicts that the 4004056 proteinis localized in the plasma membrane. SignalP predicts no known signalpeptide for this protein, however.

FIG. 9B shows the nucleotide sequence (SEQ ID NO:80) and translatedprotein sequence (SEQ ID NO:81) for clone 4004056.0.143u. Clone4004056.0.143u has an initiation codon at nucleotides 63-65 and a TGAstop codon at nucleotides 1023-1025. This sequence between residues 63and 1022 defines an ORF encoding a protein (SEQ ID NO:81) of 320 aminoacid residues (FIG. 9B).

Database searches indicate that the 4004056 protein has 306 of 311residues (98%) both identical to and positive with a 311 residue humantransmembrane domain containing protein from clone HP01862, thought tocontrol cell proliferation and differentiation. (PCT PublicationWO9927094-A2). Similarly, the protein has 306 of 311 residues (98%)identical to and positive with a 311 residue human protein (SEQ ID NO:10from PCT Publication WO9927094-A2). Clone 4004056 furthermore has 305 of311 residues (98%) both identical to and positive with the human 311residue proline-rich membrane protein (PCT Publication WO9833910-A1). Inother searches it was found that the 4004056 protein has 153 of 284residues identicdal to (53%), and 196 of 284 residues positive with(69%), the 316 residue neural membrane protein 35 (GenBank AccessionNumber AAC32463). In addition, the protein is 42% identical to, and 65%positive with, a 208 residue fragment of human NMDA receptorglutamate-binding chain (GenBank Accession Number AAB94292).

The novel 4004056 clone has a range of activities including cytokine andcell differentiation, immune stimulation/suppression, hematopoiesisregulation, tissue growth, activin/inhibin activities,chemostatic/chemokinetic activities, hemostatic/thrombolytic activities,receptor/ligand activities, tumor inhibitor, anti-inflammatory andadditional undefined activities. The 4004056 cDNAs has utility as aprobes for gene diagnosis and as gene sources for gene therapy. ThesecDNAs are also useful for large scale expression of proteins. Cellstransformed with various 4004056 nucleotides are useful for detection ofthe corresponding ligands and for screening of novel low-molecularweight pharmaceuticals.

The 4004056 protein is a likely human proline-rich membrane protein(PRMP). PRMP is similar to rat NMDA receptor glutamic acid bindingsubunit. PRMP is involved in cell signaling, protein trafficking andsubcellular localization, control of cell architecture, cell-cellinteractions, cell growth and development, and modulation of immune andinflammatory responses. The PRMP and agonists can be used to promotetissue or organ regeneration. The antagonists or inhibitors of PRMP isuseful for treating or preventing disorders associated with expressionof PRMP, e.g. inflammatory and allergic conditions such as rheumatoidand osteoarthritis, asthma, allergic rhinitis, atopic dermatitis,autoimmune conditions such as Sjogren's syndrome, scleroderma,hyperthyroidism (Grave's disease), systemic lupus, myasthenia gravis,autoimmune thyroiditis, diabetes mellitus, pancreatitis, ulcerativecolitis, Crohn's disease, atrophic gastritis, and graft versus hostdisease, disorders relating to abnormal cellular differentiation,proliferation, or degeneration, including arteriosclerosis,atherosclerosis, hyperaldosteronism, hypocortisolism (Addison'sdisease), hypothyroidism, colorectal polyps, gastric and duodenalulcers, cancers of hematopoietic cells and lymphoid tissues includingleukemias, lymphomas (including Hodgkin's disease), lymphosarcomas andmyelomas, and carcinomas of glands, tissues, and organs involved insecretion or absorption, and organs of the gastrointestinal tract.

10. Clone 4004731-1

Clone 4004731-1 is a polynucleotide (SEQ ID NO:19) comprising 1686nucleotides (FIG. 10). The clone has a Kozak sequence, an initiationcodon at positions 372-374 and a TAA termination codon at nucleotides1278-1280. The nucleotide residues between 372 and 1277 thus define anORF encoding a protein (SEQ ID NO:20) having 302 amino acid residues(FIG. 10). The PSORT predicts that the protein localizes to themitochondrial matrix space with a low certainty of 0.3600. The programSignalP predicts that no known signal peptide is present. Transcribedsequences from this gene are found in brain, pituitary, heart, breastand spleen.

In similarity searches it was found that the 4004731-1 protein has 50%identity and 67% similarity with the human laminin beta-1 chainprecursor (laminin B1 chain), a protein having 1786 residues (GenBankAccession Number P07942). Laminins are a major component of the basementmembrane and have several biologically active sites that regulateangiogenesis and tumor growth. Grant et al., 1994 Pathol Res Pract.190(9-10): 854-863. Laminins strongly stimulate axon outgrowth in vitro,and are transiently expressed in embryonic development and after CNSinjury. Luebke et al., 1995 J. Neurobiol 27(1): 1-14. In addition,Laminin BI expression is greatly disturbed in severely diseased patientswith severe childhood autosomal recessive muscular dystrophy. Yamada etal., 1995 Lab Invest. 72(6): 715-722. Clone 4004731-1 thus hasdiagnostic and therapeutic utility in pathologies related to musculardystrophy, cell outgrowth, cell proliferation, angiogenesis, and neuraldevelopment and in CNS pathologies, e.g., CNS injury, Alzheimer'sdisease and parkinsonism.

11. Clone 4009334-1

Clone 4009334-1 includes a polynucleotide (SEQ ID NO:21) having 2010nucleotides (FIG. 11). This clone is similar to clone 4009334-2 (below),but is longer than the latter because of inserts at nucleotides1361-1440 and 1541-1597 in SEQ ID NO:21. These differences are thoughtto arise from splicing variations of the mRNA. Clone 4009334-1 has astart codon at positions 243-245 and a TGA termination codon atnucleotides 1659-1661. Residues between 243 and 1658 therefore define anORF encoding a protein (SEQ ID NO:22) of 472 residues (FIG. 11). ThePSORT software program affords a weak prediction that the 4009334-1protein localizes to the microbody (certainty=0.30). The SignalPsoftware program predicts that the protein lacks a known signal peptide.Transcribed sequences from this gene are found in OVCAR-3 cells, MCF-7cells, mammary gland, lung, including fetal lung, brain, includingthalamus, adrenal gland, salivary gland, pancreas, heart, white bloodcells and Raji cells.

The 4009334-1 protein has 272 of 304 residues (89%) identical to, and278 of 304 residues (91%) positive with, the 550 residue AVENA proteinfrom Gallus gallus (chicken) (GenBank Accession Numbers AB017437 andBAA33016). It also has 218 of 251 residues (86%) identical to, and 228of 251 residues (90%) positive with, a murine 783 residue enabledhomolog (neural variant MENA+ protein) (GenBank Accession NumberAAC52864). The 4009334-1 protein additionally has 94 of 146 residues(64%) identical to, and 107 of 146 residues (73%) positive with, the 380residue human vasodilator-stimulated phosphoprotein (VASP) (GenBankAccession Number P50552).

Clone 4009334-1 thus has diagnostic and therapeutic utility inpathologies related to cellular control, cell proliferation, celldevelopment and cell differentiation. Clone 4009334-1 is also useful inangiogenesis, carcinogenesis, and in body and organ homeostasis.

12. Clone 4009334-2

Clone 4009334-2 includes a polynucleotide (SEQ ID NO:23) of 1952nucleotides (FIG. 12) that appears to be a shorter splice variant ofclone 4009334-1 (SEQ ID NO:21, above). The 4009334-2 nucleic acidsequence includes a Kozak sequence, an initiation codon at nucleotides243-245 and a stop codon at positions 1716-1718. Residues between 243and 1715 define an ORF encoding a protein (SEQ ID NO:24) having 491amino acid residues (FIG. 12). The PSORT program affords a weakprediction that the 4009334-1 protein localizes to the microbody(certainty=0.30). The SignalP program predicts that the protein lacks asignal peptide. Transcribed sequences from this gene are found inOVCAR-3 cells, MCF-7 cells, mammary gland, lung, including fetal lung,brain, including thalamus, adrenal gland, salivary gland, pancreas,heart, white blood cells and Raji cells.

The 4009334-2 protein has 272 of 304 residues (89%) identical to, and278 of 304 residues (91%) positive with, the 550 residue d1033982(GenBank Accession Number AB017437) AVENA protein from Gallus gallus(chicken) (GenBank Accession Number BAA33016). It also has 218 of 251residues (86%) identical to, and 228 of 251 residues (90%) positivewith, a murine 783 residue enabled homolog (neural variant MENA+protein) (GenBank Accession Number AAC52864). The 4009334-2 proteinadditionally has 94 of 146 residues (64%) identical to, and 107 of 146residues (73%) positive with, the 380 residue humanvasodilator-stimulated phosphoprotein (VASP) (GenBank Accession NumberP50552).

Clone 4009334-2 thus has diagnostic and therapeutic utility inpathologies related to cellular control, cell proliferation, celldevelopment and cell differentiation. Clone 4009334-2 is also useful inangiogenesis, carcinogenesis, pathologies related to neoplasia, and bodyand organ homeostasis.

13. Clone 4035508

Clone 4035508 includes a polynucleotide sequence (SEQ ID NO:25) of 827nucleotides (FIG. 13). The clone includes a Kozak sequence, a startcodon at positions 233-235 and a TGA stop codon at nucleotides 602-604,thus setting forth an ORF between residues 233 and 601 encoding apolypeptide (SEQ ID NO:26) having 123 residues (FIG. 13). The PSORTprogram predicts that the 4035508 protein localizes to the plasmamembrane. The SignalP program predicts that the 4035508 polypeptide hasa signal peptide whose most probable cleavage site occurs betweenresidues 29 and 30, represented by the dash between the amino acidsLFG-WP (i.e., LeuPheGly-TrpPro). Transcribed sequences from this geneare found in fetal lung tissue, and in multiple adult tissue types,including lymph node tissues.

Similarity searching reveals that the 4035508 protein has 37 of 108residues (34%) identical to, and 55 of 108 residues (50%) positive with,a 559 residue human protein PB39 (POV1; GenBank Accession NumberAAC33004), a predicted secreted protein upregulated and alternativelyspliced in prostate cancer. Cole et al., 1998 Genomics 51(2): 282-287.

PB39 plays a role in the development of human prostate cancer. Byanalogy, successful therapeutic targeting of 4035508 and/or its splicevariants to a mammalian subject will result in provide significantanti-tumor activity, especially in combination with establishedcytotoxic/genotoxic therapies (i.e. chemosensitization andradiosensitization). Moreover successful therapeutic targeting of4035508 will also limit the extent, frequency, or both of metastaticdissemination (tumor burden). 4035508 will also limit tumor angiogenesissince 4035508 is highly expressed in activated endothelial cells, suchas human umbilical vein endothelial cells (HUVECs).

Therapeutic targeting of 4035508 and its splice variants will also beprovided via generation of human or humanized monoclonal antibodies thatblock the ability of 4035508 to interact with cognate receptor(s) andelicit a transmembrane signal(s). Equally, the generation of smallmolecules (synthetics, cell permeable peptides, other) than specificallyinterfere with one or more of the downstream signaling components in thepathway(s) activated by 4035508 bound to cognate receptor(s) will havesignificant anti-tumor activity as described above. Likewise, theintroduction of antisense constructs (naked DNA, adenoviral constructs),ribozymes to inhibit the expression of 4035508 in select humanmalignancies will have significant anti-tumor activity as describedabove.

Clone 4035508 has diagnostic and therapeutic utility in pathologiesrelated to neoplasias, cell proliferation and cellular control. Based onthe expression profile of the 4035508 gene (see Example 10 and FIG. 22)the therapeutic indications for targeting 4035508 include metastaticcolon carcinomas (up regulation in SW620 metastatic variant of SW480),breast adenocarcinomas, glioma/astrocytomas, small cell lung cancers andmalignant melanomas.

14. Clone 4339264

Clone 4339264 includes a polynucleotide (SEQ ID NO:27) of 1063nucleotides (FIG. 14). The clone includes an initiation codon atpositions 48-50 and TAA termination codon at positions 945-947. Thisclone includes an ORF from residues 48 to 944 encoding a protein (SEQ IDNO:28) of 299 amino acid residues (FIG. 14). The PSORT program predictsthat the 4339264 protein localizes in the plasma membrane with acertainty of 0.6000. The SignalP program predicts that there is a signalpeptide whose cleavage site most likely occurs between residues 69 and70, represented by the dash between the amino acids LQA-RF (i.e.,LeuGlnAla-ArgPhe). The clone originates in DNA isolated from lymph node.Transcribed sequences from this gene are found in MCF-7 cells, OVCAR-3cells, heart, prostate, uterus, mammary gland, salivary gland, thalamus,bone marrow, lymph node, spleen, fetal liver, fetal thymus-CRL7046, and10 human total RNA libraries from Clontech, Inc. (Palo Alto, Calif.;brain, fetal brain, liver, fetal liver, skeletal muscle, pancreas,kidney, heart, lung and placenta).

In a similarity search, it was found that the 4339264 protein has 194 of219 residues (88%) identical to, and 207 of 219 residues (94%) positivewith the 296 residue myeloid upregulated protein of mouse (GenBankAccession Number 035682). In addition, the protein has 39 of 125residues (31%) identical to, and 58 of 125 residues (46%) positive withthe 153 residue human four transmembrane domain MAL T-lymphocytematuration-associated protein (GenBank Accession Number P21145). The MALprotein is believed to act as a signaling receptor and transporter ofwater-soluble molecules and ions across the lipid bilayer. Alonso andWeissman 1987 Proc Natl Acad Sci U.S.A. 84(7): 1997-2000.

The breadth of expression of 4339264 transcript among a wide range ofnormal and cancerous tissues identified using quantitative real-time PCR(Example 11 and FIG. 23) suggests that the protein encoded by the4339264 gene has a generalized role in cell homeostasis. Expression of4339264 is elevated in select human cancer cell lines relative to thetissue of origin and elevated in some fetal tissue relative to the adulttissue, indicating a role in organogenesis and tissue repair.Overexpression of 4339264 should therefore contribute to tumor genesis.In addition, high expression of 4339264 in fetal kidney relative to theadult kidney suggests a likely role of 4339264 in organogenesis.

Successful therapeutic targeting and downregulation of 4339264 and/orits splice variants will result in significant anti-tumor activity,especially in combination with established cytotoxic/genotoxic therapies(i.e. chemosensitization, radiosensitization). Moreover successfultherapeutic targeting of 4339264 will also limit the extent andfrequency of metastatic dissemination (i.e., tumor burden) andpotentially limit tumor angiogenesis.

Therapeutic targeting of 4339264 and its splice variants will also beprovided by generation of human or humanized monoclonal antibodies thatblock the ability of 4339264 to interact with cognate receptor(s) andelicit a transmembrane signal(s). Equally, the generation of smallmolecules (i.e., synthetics, cell permeable peptides, other) thatspecifically interfere with one or more of the downstream signalingcomponents in the pathway(s) activated by 4339264 when bound to specificcognate receptor(s) will have significant anti-tumor activity asdescribed above. Likewise, the introduction of antisense constructs(i.e., naked DNA, adenoviral constructs) or ribozymes to inhibit theexpression of 4339264 in select human malignancies will also havesignificant anti-tumor activity as described above.

Clone 4339264 thus has diagnostic and therapeutic utility in pathologiesrelated to cell signaling, regulation and development. Based on theexpression profile of the 4339264 gene (Example 11 and FIG. 23) thetherapeutic indications for targeting 4339264 include malignantmelanomas, small cell lung carcinomas and renal cell carcinoma

Nucleic Acids

One aspect of the invention pertains to isolated nucleic acid molecules(i.e., SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74,76, 78 and 80, plus 29 and 31) that encode the SECX polypeptides of theinvention, wherein the SECX polypeptides are selected from the groupcomprising clone 2777610, clone 2864933-1, clone 2864933-2, clone2982339, clone 3352358-1, clone 3352358-2, clone 3884846, clone3884846-1, clone 3884846-2, clone 3911675, clone 3911675-2, clone4004056, clone 4004056.0.143u, clone 4004731-1, clone 4009334-1, clone4009334-2, clone 4035508, and clone 4339264 polypeptides, (i.e., SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 75, 77, 79 and81, plus 30 and 32; see Table 1 and FIGS. 1-17), or biologically activeportions thereof, as well as nucleic acid fragments sufficient for useas hybridization probes to identify SECX-encoding nucleic acids (e.g.,SECX mRNA) and fragments for use as PCR primers for the amplification ormutation of SECX nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs, and derivatives, fragments andhomologs thereof. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. SECX nucleicacids of the invention include SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and 31 (Table 1 and FIGS.1-17), and fragments, homologs, and derivatives thereof.

“Probes” refer to nucleic acid sequences of variable length, preferablybetween at least about 10 nucleotides (nt), 100 nt, or as many as about,e.g., 6,000 nt, depending on use. Probes are used in the detection ofidentical, similar, or complementary nucleic acid sequences. Longerlength probes are usually obtained from a natural or recombinant source,are highly specific and much slower to hybridize than oligomers. Probesmay be single- or double-stranded and designed to have specificity inPCR, membrane-based hybridization technologies, or ELISA-liketechnologies.

An “isolated” nucleic acid molecule is one that is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, an isolated nucleic acid molecule encoding any oneof the SECX polypeptides, including chemokine receptor-like protein,semaphorin protein-like splice variants, a putative mitochondrialprotein (clone 2982339), SLIT protein-like splice variants, a putativemicrobody (peroxisome) associated protein (clone 3884846), atetraspanin-like protein, a putative proline-rich membrane protein(clone 4004056), a laminin β-chain precursor-like protein, AVENAprotein-like splice variants (clones 4009334-1 and 4009334-2), a fetallung-associated protein (clone 4035508) and a myeloid upregulatedprotein (clone 4339264), can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived (e.g., adult and fetal cells from tissuesincluding bone tissue (including bone marrow), heart, lymph node,pancreas, spleen, thymus, placenta, kidney, liver, thalamus, brain,pituitary, breast, lung, salivary gland and adrenal gland). Moreover, an“isolated” nucleic acid molecule, e.g., a cDNA molecule, can besubstantially free of other cellular material or culture medium whenproduced by recombinant techniques, or of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a SECX nucleicacid molecule having the nucleotide sequence of SEQ ID NOs:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, plus 29 or 31, or a complementof any of these nucleotide sequences, can be isolated using standardmolecular biology techniques and the sequence information providedherein. Using all or a portion of the SECX nucleic acid sequences of SEQID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, plus 29 or31, or a complement of any of these nucleotide sequences, as ahybridization probe, said SECX molecules can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook etal., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2^(nd) Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; andAusubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley & Sons, New York, N.Y., 1993.)

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to SECX nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm, or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence havingabout 10 nt, 50 nt, or 100 nucleotides in length, preferably about 15nucleotides to 30 nucleotides in length. In one embodiment, anoligonucleotide comprising a nucleic acid molecule less than 100nucleotides in length would further comprise at lease 6 contiguousnucleotides of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 74, 76, 78 and 80, plus 29 and 31, or a complement thereof.Oligonucleotides may be chemically synthesized and may be used asprobes.

In an embodiment, an isolated nucleic acid molecule of the inventioncomprises a SECX nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and 31, or a portion ofthis nucleotide sequence. A nucleic acid molecule that is complementaryto said SECX nucleotide sequences is one that is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NOs:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and31, or a portion of this nucleotide sequence, that it can hydrogen bondwith little or no mismatches to the given SECX nucleotide sequence,thereby forming a stable duplex.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotides units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, Von der Waals, hydrophobic interactions, etc. Aphysical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and 31, orthe nucleotide sequence of the DNA insert of the plasmid, e.g., e.g.,the pSecTag2 B and pSecV5His vectors described in Example 3, whereine.g., a fragment that can be used as a probe or primer or a fragmentencoding a biologically active portion of SECX. Fragments providedherein are defined as sequences of at least 6 (contiguous) nucleic acidsor at least 4 (contiguous) amino acids, a length sufficient to allow forspecific hybridization in the case of nucleic acids or for specificrecognition of an epitope in the case of amino acids, respectively, andare at most some portion less than a full length sequence. Fragments maybe derived from any contiguous portion of a nucleic acid or amino acidsequence of choice. Derivatives are nucleic acid sequences or amino acidsequences formed from the native compounds either directly or bymodification or partial substitution. Analogs are nucleic acid sequencesor amino acid sequences that have a structure similar to, but notidentical to, the native compound but differs from it in respect tocertain components or side chains. Analogs may be synthetic or from adifferent evolutionary origin and may have a similar or oppositemetabolic activity compared to wild type. Homologs are nucleic acidsequences or amino acid sequences of a particular gene that are derivedfrom different species.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 30%, 50%, 70%, 80%, or 95% identity (with a preferred identity of80-95%) over a nucleic acid or amino acid sequence of identical size orwhen compared to an aligned sequence in which the alignment is done by acomputer homology program known in the art (e.g., see below), or whoseencoding nucleic acid is capable of hybridizing to the complement of asequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. See e.g. Ausubel, etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993, and below.

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of SECX polypeptide. Isoforms can be expressed in differenttissues of the same organism as a result of, for example, alternativesplicing of RNA. Alternatively, isoforms can be encoded by differentgenes. In the present invention, homologous nucleotide sequences includenucleotide sequences encoding for a SECX polypeptide of species otherthan humans, including, but not limited to, mammals, and thus caninclude, e.g., mouse, rat, rabbit, dog, cat cow, horse, and otherorganisms. Homologous nucleotide sequences also include, but are notlimited to, naturally occurring allelic variations and mutations of thenucleotide sequences set forth herein. A homologous nucleotide sequencedoes not, however, include the nucleotide sequence encoding human SECXprotein. Homologous nucleic acid sequences include those nucleic acidsequences that encode conservative amino acid substitutions (see below)in SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76,78 and 80, plus 29 and 31, as well as a polypeptide having SECXactivity. Biological activities of the individual SECX proteins aredescribed above. A homologous amino acid sequence does not encode theamino acid sequence of a human SECX polypeptide.

A SECX polypeptide is encoded by the open reading frame (“ORF”) of aSECX nucleic acid. The invention includes the nucleic acid sequencecomprising the stretch of nucleic acid sequences of SEQ ID NOs:1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and31, that comprises the ORF of that nucleic acid sequence and encodes apolypeptide of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 75, 77, 79 and 81, plus 30 and 32.

An “open reading frame” (“ORF”) corresponds to a nucleotide sequencethat could potentially be translated into a polypeptide. A stretch ofnucleic acids comprising an ORF is uninterrupted by a stop codon. An ORFthat represents the coding sequence for a full protein begins with anATG “start” codon and terminates with one of the three “stop” codons,namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF maybe any part of a coding sequence, with or without a start codon, a stopcodon, or both. For an ORF to be considered as a good candidate forcoding for a bona fide cellular protein, a minimum size requirement isoften set, for example, a stretch of DNA that would encode a protein of50 amino acids or more.

The nucleotide sequence determined from the cloning of the human SECXgene allows for the generation of probes and primers designed for use inidentifying and/or cloning SECX homologues in other cell types, e.g.from other tissues, as well as SECX homologues from other mammals. Theprobe/primer typically comprises substantially purified oligonucleotide.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12, 25, 50,100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotidesequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 74, 76, 78 and 80, plus 29 and 31, or the nucleotide sequence of theDNA insert of the plasmid such as, e.g., the pSecTag2 B and pSecV5Hisvectors described in Example 3; or an anti-sense strand nucleotidesequence of a SECX nucleotide or the anti-sense strand SECX nucleotidesequence of the DNA insert of the plasmid known in the art; or of anaturally occurring mutant of a SECX nucleotide, or the naturallyoccurring mutant of the DNA insert of the plasmid vector known in theart.

Probes based on the human SECX nucleotide sequence can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In various embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a SECX protein, e.g., by measuring a level of aSECX-encoding nucleic acid in a sample of cells from a subject e.g.,detecting SECX mRNA levels or determining whether a genomic SECX genehas been mutated or deleted.

“A polypeptide having a biologically active portion of SECX” refers topolypeptides exhibiting activity similar, but not necessarily identicalto, an activity of a polypeptide of the present invention, includingmature forms, as measured in a particular biological assay, with orwithout dose dependency. A nucleic acid fragment encoding a“biologically active portion of SECX” can be prepared by isolating aportion of a SECX nucleotide that encodes a polypeptide having a SECXbiological activity (wherein the biological activities of the SECXproteins are described above), expressing the encoded portion of SECXprotein (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of SECX. For example, a nucleic acidfragment encoding a biologically active portion of SECX includes anextracellular domain, e.g., the clone 2864933-1 amino acid residues 19to 644 of SEQ ID NO:4. In another embodiment, a nucleic acid fragmentencoding a biologically active portion of SECX that includes anextracellular domain includes the DNA encoding such domains, e.g., atleast the nucleic acids of SEQ ID NO:9 that encodes the human clone3352358-1 extracellular domain represented by amino acid residues 42 to486 of SEQ ID NO:10.

SECX Variants

The invention further encompasses any one or more nucleic acid moleculesthat differ from the SECX nucleotide sequence shown in at least one ofSEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78and 80, plus 29 and 31, due to degeneracy of the genetic code and thusencode the same SECX protein as that encoded by any of the abovenucleotide sequences. In another embodiment, an isolated SECX nucleicacid molecule of the invention has a nucleotide sequence encoding aprotein having any one amino acid sequence shown in SEQ ID NOs:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 75, 77, 79 and 81, plus 30and 32.

In addition to these human SECX nucleotide sequences, or the SECXnucleotide sequence of the DNA insert of a plasmid or vector, it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to changes in the amino acid sequences of a SECX may existwithin a population (e.g., the human population). Such geneticpolymorphism in a SECX gene may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules comprisingan open reading frame encoding a SECX protein, preferably a mammalianSECX protein. Such natural allelic variations can typically result in1-5% variance in the nucleotide sequence of the SECX gene. Any and allsuch nucleotide variations and resulting amino acid polymorphisms inSECX that are the result of natural allelic variation and that do notalter the functional activity of SECX are intended to be within thescope of the invention.

Moreover, nucleic acid molecules encoding SECX proteins from otherspecies, and thus that have a nucleotide sequence that differs from thehuman sequence disclosed herein, are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelicvariants and homologues of a SECX cDNAs of the invention can be isolatedbased on their homology to the human SECX nucleic acids disclosed hereinusing the human cDNAs, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. For example, a soluble human SECX cDNA can beisolated based on its homology to human membrane-bound SECX. Likewise, amembrane-bound human SECX cDNA can be isolated based on its homology tosoluble human SECX.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising at leastone SECX nucleotide sequence. In another embodiment, the nucleic acid isat least 10, 25, 50, 100, 250, 500 or 2000 nucleotides in length. Inanother embodiment, an isolated nucleic acid molecule of the inventionhybridizes to the coding region. As used herein, the term “hybridizesunder stringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.

Homologs (i.e., nucleic acids encoding SECX proteins derived fromspecies other than human) or other related sequences (e.g., paralogs)can be obtained by low, moderate or high stringency hybridization withall or a portion of the particular human sequence as a probe usingmethods well known in the art for nucleic acid hybridization andcloning.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a probe, primer or oligonucleotide willhybridize to its target sequence, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures than shorter sequences. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

Stringent conditions are known to those skilled in the art and can befound in Ausubel et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditionsare such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%,98%, or 99% homologous to each other typically remain hybridized to eachother. A non-limiting example of stringent hybridization conditions arehybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/mldenatured salmon sperm DNA at 65° C., followed by one or more washes in0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of theinvention that hybridizes under stringent conditions to a SECXnucleotide sequence corresponds to a naturally-occurring nucleic acidmolecule. As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable toat least one SECX nucleic acid molecule, or fragments, analogs orderivatives thereof, under conditions of moderate stringency isprovided. A non-limiting example of moderate stringency hybridizationconditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDSand 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one ormore washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderatestringency that may be used are well-known in the art. See, e.g.,Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION,A LABORATORY MANUAL, Stockton Press, N.Y.

In a third embodiment, a nucleic acid that is hybridizable to at leastone SECX nucleic acid molecule, or fragments, analogs or derivativesthereof, under conditions of low stringency, is provided. A non-limitingexample of low stringency hybridization conditions are hybridization in35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%(wt/vol) dextran sulfate at 40° C., followed by one or more washes in2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Otherconditions of low stringency that may be used are well known in the art(e.g., as employed for cross-species hybridizations). See, e.g., Ausubelet al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, ALABORATORY MANUAL, Stockton Press, N.Y.; Shilo and Weinberg, 1981, ProcNatl Acad Sci USA 78: 6789-6792.

Conservative Mutations

In addition to naturally-occurring allelic variants of the SECX sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced by mutation into at least oneSECX nucleotide sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and 31, thereby leadingto changes in the amino acid sequence of the encoded SECX protein,without altering the functional ability of the SECX protein. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 75, 77, 79and 81, plus 30 and 32, or the SECX nucleotide sequence of the DNAinsert of the plasmid or vector known in the art. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of SECX without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the SECX proteinsof the present invention, are predicted to be particularly unamenable toalteration.

Another aspect of the invention pertains to nucleic acid moleculesencoding SECX proteins that contain changes in amino acid residues thatare not essential for activity. Such SECX proteins differ in amino acidsequence from SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 75, 77, 79 and 81, plus 30 and 32, yet retain biological activity.In one embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a protein, wherein the protein comprises anamino acid sequence at least about 45% homologous to at least one SECXamino acid sequence. Preferably, the protein encoded by the nucleic acidmolecule is at least about 60% homologous to at least one SECXpolypeptide, more preferably at least about 70% homologous, at leastabout 80% homologous, at least about 90% homologous, and most preferablyat least about 95% homologous to that given SECX polypeptide.

An isolated nucleic acid molecule encoding a SECX protein homologous toa given SECX protein can be created by introducing one or morenucleotide substitutions, additions or deletions into the correspondingSECX nucleotide sequence, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein.

Mutations can be introduced into SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and 31, by standardtechniques, e.g., site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in SECX is replaced withanother amino acid residue from the same side chain family.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a SECX coding sequence, e.g., bysaturation mutagenesis, and the resultant mutants can be screened forSECX biological activity to identify mutants that retain activity.Following mutagenesis, the encoded SECX protein can be expressed by anyrecombinant technology known in the art and the activity of the proteincan be determined.

In one embodiment, a mutant SECX protein can be assayed for (1) theability to form protein:protein interactions with other SECX proteins,other cell-surface proteins, or biologically active portions thereof,(2) complex formation between a mutant SECX protein and a SECX ligand;(3) the ability of a mutant SECX protein to bind to an intracellulartarget protein or biologically active portion thereof; (e.g. avidinproteins).

Antisense

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that are hybridizable to or complementary to a SECXnucleic acid molecule, or fragments, analogs or derivatives thereof. An“antisense” nucleic acid comprises a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. In specific aspects, antisensenucleic acid molecules are provided that comprise a sequencecomplementary to at least about 10, 25, 50, 100, 250 or 500 nucleotidesor an entire SECX coding strand, or to only a portion thereof. Nucleicacid molecules encoding fragments, homologs, derivatives and analogs ofa SECX protein, or antisense nucleic acids complementary to a SECXnucleic acid sequence, are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingSECX. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., ORFs shown in FIGS. 1-17). In another embodiment, the antisensenucleic acid molecule is antisense to a “noncoding region” of the codingstrand of a nucleotide sequence encoding SECX. The term “noncodingregion” refers to 5′ and 3′ sequences which flank the coding region thatare not translated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding SECX disclosed herein (e.g.,SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78and 80, plus 29 and 31), antisense nucleic acids of the invention can bedesigned according to the rules of Watson and Crick or Hoogsteen basepairing. The antisense nucleic acid molecule can be complementary to theentire coding region of SECX mRNA, but more preferably is anoligonucleotide that is antisense to only a portion of the coding ornoncoding region of SECX mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of SECX mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis or enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a SECX proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies that bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett215: 327-330).

Ribozymes and PNA Moieties

Nucleic acid modifications include, by way of nonlimiting example,modified bases, and nucleic acids whose sugar phosphate backbones aremodified or derivatized. These modifications are carried out at least inpart to enhance the chemical stability of the modified nucleic acid,such that they may be used, for example, as antisense binding nucleicacids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,e.g., an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveSECX mRNA transcripts to thereby inhibit translation of SECX mRNA. Aribozyme having specificity for a SECX-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a SECX cDNA disclosedherein (i.e., SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 74, 76, 78 and 80, plus 29 and 31). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a SECX-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,SECX mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel etal., (1993) Science 261:1411-1418.

Alternatively, SECX gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a SECXgene (e.g., the SECX promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the SECX gene in target cells.See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. etal. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14: 807-15.

In various embodiments, the nucleic acids of SECX can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg MedChem 4: 5-23). As used herein, the terms “peptide nucleic acids” or“PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al. (1996) above; Perry-O'Keefe etal. (1996) PNAS 93: 14670-675.

PNAs of SECX can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofSECX can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNAsequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe(1996), above).

In another embodiment, PNAs of SECX can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of SECX can be generated that may combinethe advantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNase H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996) above). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupledin a stepwise manner to produce a chimeric molecule with a 5′ PNAsegment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization triggered cleavage agents (See, e.g., Krolet al., 1988, BioTechniques 6:958-976) or intercalating agents. (See,e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, etc.

SECX Proteins

The novel protein of the invention includes the SECX proteins whosesequences are provided in FIGS. 1-15 and 17 (SEQ ID NOs:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 75, 77, 79 and 81, plus 30 and 32).The invention also includes a mutant or variant protein, any of whoseresidues may be changed from the corresponding residue shown in FIGS.1-15 and 17 while still encoding a protein that maintains its SECXactivities and physiological functions, or a functional fragmentthereof. In the mutant or variant protein, up to 20% or more of theresidues may be so changed.

In general, an SECX variant that preserves SECX-like function includesany variant in which residues at a particular position in the sequencehave been substituted by other amino acids, and further include thepossibility of inserting an additional residue or residues between tworesidues of the parent protein as well as the possibility of deletingone or more residues from the parent sequence. Any amino acidsubstitution, insertion, or deletion is encompassed by the invention. Infavorable circumstances, the substitution is a conservative substitutionas defined above.

One aspect of the invention pertains to isolated SECX proteins, andbiologically active portions thereof, or derivatives, fragments, analogsor homologs thereof. Also provided are polypeptide fragments suitablefor use as immunogens to raise anti-SECX antibodies. In one embodiment,native SECX proteins can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, SECX proteins are produced byrecombinant DNA techniques. Alternative to recombinant expression, aSECX protein or polypeptide can be synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theSECX protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of SECXprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of SECX protein having less than about 30% (by dryweight) of non-SECX protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-SECX protein,still more preferably less than about 10% of non-SECX protein, and mostpreferably less than about 5% non-SECX protein. When the SECX protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of SECX protein in which the protein isseparated from chemical precursors or other chemicals that are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of SECX protein having less than about 30% (by dry weight)of chemical precursors or non-SECX chemicals, more preferably less thanabout 20% chemical precursors or non-SECX chemicals, still morepreferably less than about 10% chemical precursors or non-SECXchemicals, and most preferably less than about 5% chemical precursors ornon-SECX chemicals.

Biologically active portions of a SECX protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the SECX protein, e.g., the amino acidsequence shown in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 75, 77, 79 and 81, plus 30 and 32, that include fewer aminoacids than the full length SECX proteins, and exhibit at least oneactivity of a SECX protein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the SECXprotein. A biologically active portion of a SECX protein can be apolypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length.

It is to be understood that a biologically active portion of a SECXprotein of the present invention may contain at least one of thestructural domains identified in Sections 1-14, above. An alternativebiologically active portion of a SECX protein may contain anextracellular domain of the SECX protein. Another biologically activeportion of a SECX protein may contain the transmembrane domain of theSECX protein. Yet another biologically active portion of a SECX proteinof the present invention may contain the intracellular domain of theSECX protein.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native SECXprotein.

In an embodiment, the SECX protein has any one or more amino acidsequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 75, 77, 79 and 81, plus 30 and 32. In other embodiments, theSECX protein is substantially homologous to any one of SEQ ID NOs:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 75, 77, 79 and 81, plus 30and 32, and retains the functional activity of that given SECX proteinyet differs in amino acid sequence due to natural allelic variation ormutagenesis, as described in detail below. Accordingly, in anotherembodiment, the SECX protein is a protein that comprises an amino acidsequence at least about 75% homologous to any one amino acid sequence ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 75, 77,79 and 81, plus 30 and 32, and retains the functional activity of thatSECX protein.

This invention further features isolated SECX protein, or derivatives,fragments, analogs or homologs thereof, that is encoded by a nucleicacid molecule having a nucleotide sequence that hybridizes understringent hybridization conditions to a nucleic acid molecule comprisingthe nucleotide sequence of any one or more of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78 and 80, plus 29 and 31.

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48:443-453. Using GCG GAP software with the following settings for nucleicacid sequence comparison: GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3, the coding region of the analogous nucleic acidsequences referred to above exhibits a degree of identity preferably ofat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (i.e.,encoding) part of the DNA sequence shown in any one or more of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78 and80, plus 29 and 31.

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region. Similar calculation are used when comparingamino acid residues in polypeptide sequences.

Chimeric and Fusion Proteins

The invention also provides SECX chimeric or fusion proteins. As usedherein, a SECX “chimeric protein” or “fusion protein” comprises a SECXpolypeptide operatively linked to a non-SECX polypeptide. A “SECXpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to SECX, whereas a “non-SECX polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinthat is not substantially homologous to the SECX protein, e.g., aprotein that is different from the SECX protein and that is derived fromthe same or a different organism. Within a SECX fusion protein the SECXpolypeptide can correspond to all or a portion of a SECX protein. In oneembodiment, a SECX fusion protein comprises at least one biologicallyactive portion of a SECX protein. In another embodiment, a SECX fusionprotein comprises at least two biologically active portions of a SECXprotein. In yet another embodiment, a SECX fusion protein comprises atleast three biologically active portions of a SECX protein. Within thefusion protein, the term “operatively linked” is intended to indicatethat the SECX polypeptide and the non-SECX polypeptide are fusedin-frame to each other. The non-SECX polypeptide can be fused to theN-terminus or C-terminus of the SECX polypeptide.

For example, in one embodiment a SECX fusion protein comprises a SECXdomain operably linked to the extracellular domain of a second proteinknown to be involved in an activity of interest. Such fusion proteinscan be further utilized in screening assays for compounds which modulateSECX activity (such assays are described in detail below).

In one embodiment, the fusion protein is a GST-SECX fusion protein inwhich the SECX sequences are fused to the C-terminus of the GST (i.e.,glutathione S-transferase) sequences. Such fusion proteins canfacilitate the purification of recombinant SECX.

In another embodiment, the fusion protein is a SECX protein containing aheterologous signal sequence at its N-terminus. For example, the nativeSECX signal sequence (i.e., about amino acids 1 to 26, or as describedin Sections 1-14 above) can be removed and replaced with a signalsequence from another protein. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of SECX can be increasedthrough use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a SECX-immunoglobulinfusion protein in which the SECX sequences comprising primarily theextracellular domains are fused to sequences derived from a member ofthe immunoglobulin protein family. The SECX-immunoglobulin fusionproteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween a SECX ligand and a SECX protein on the surface of a cell, tothereby suppress SECX-mediated signal transduction in vivo. TheSECX-immunoglobulin fusion proteins can be used to affect thebioavailability of a SECX cognate ligand. Inhibition of the SECXligand/SECX interaction may be useful therapeutically for both thetreatment of proliferative and differentiative disorders, as well asmodulating (e.g. promoting or inhibiting) cell survival. Moreover, theSECX-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-SECX antibodies in a subject, to purify SECXligands, and in screening assays to identify molecules that inhibit theinteraction of SECX with a SECX ligand.

A SECX chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A SECX-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theSECX protein.

The invention also provides signal sequences derived from various SECXpolypeptides. The signal sequences include, e.g., polypeptides includingthe signal peptides identified for the SECX polypeptides as predicted bythe SignalP software program for the SECX polypeptides described above.These signal sequences are useful for directing a linked polypeptidesequence to a desired intracellular or extracellular (if secretion fromthe cell is desired) location. In some embodiments, the signal sequenceincludes a portion of a SECX signal sequence that is sufficient todirect a linked polypeptide to a desired cellular compartment.

SECX Agonists and Antagonists

The present invention also pertains to variants of the SECX proteinsthat function as either SECX agonists (mimetics) or as SECX antagonists.Variants of the SECX protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the SECX protein. An agonist ofthe SECX protein can retain substantially the same, or a subset of, thebiological activities of the naturally occurring form of the SECXprotein. An antagonist of the SECX protein can inhibit one or more ofthe activities of the naturally occurring form of the SECX protein by,for example, competitively binding to a downstream or upstream member ofa cellular signaling cascade which includes the SECX protein. Thus,specific biological effects can be elicited by treatment with a variantof limited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the SECXproteins.

Variants of the SECX protein that function as either SECX agonists(mimetics) or as SECX antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theSECX protein for SECX protein agonist or antagonist activity. In oneembodiment, a variegated library of SECX variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of SECX variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential SECX sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of SECX sequences therein. There are avariety of methods which can be used to produce libraries of potentialSECX variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential SECX sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.

Polypeptide Libraries

In addition, libraries of fragments of the SECX protein coding sequencecan be used to generate a variegated population of SECX fragments forscreening and subsequent selection of variants of a SECX protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a SECX coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA that can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the SECX protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of SECX proteins. The mostwidely used techniques, which are amenable to high throughput analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify SECX variants (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6:327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated SECX library, e.g., a library of mutant SECX polypeptides.For example, a library of expression vectors can be transfected into acell line that ordinarily responds to a particular ligand or receptor ina SECX-dependent manner, e.g., through a signaling complex. Thetransfected cells are then contacted with the putative SECX interactantand the effect of expression of the mutant SECX on signaling by thesignaling complex can be detected, e.g. by measuring a cellular activityor cell survival. Plasmid DNA can then be recovered from the cells whichscore for inhibition, or alternatively, potentiation of, e.g., cytokineinduction, and the individual clones further characterized.

Anti-SECX Antibodies

The invention encompasses antibodies and antibody fragments, such asF_(ab) or (F_(ab))₂, that bind immunospecifically to any of thepolypeptides of the invention.

An isolated SECX protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind SECX using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length SECX protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of SECX for use as immunogens. Theantigenic peptide of SECX comprises at least 4 amino acid residues ofthe amino acid sequence shown in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 75, 77, 79 and 81, plus 30 and 32 andencompasses an epitope of SECX such that an antibody raised against thepeptide forms a specific immune complex with SECX. Preferably, theantigenic peptide comprises at least 6, 8, 10, 15, 20, or 30 amino acidresidues. Longer antigenic peptides are sometimes preferable overshorter antigenic peptides, depending on use and according to methodswell known to someone skilled in the art.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of SECX that is locatedon the surface of the protein, e.g. a hydrophilic region. Ahydrophobicity analysis of the human SECX protein sequence will indicatewhich regions of a SECX polypeptide are particularly hydrophilic and,therefore, are likely to encode surface residues useful for targetingantibody production. As a means for targeting antibody production,hydropathy plots showing regions of hydrophilicity and hydrophobicitymay be generated by any method well known in the art, including, forexample, the Kyte Doolittle or the Hopp Woods methods, either with orwithout Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc.Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.Biol. 157: 105-142, each incorporated herein by reference in theirentirety.

As disclosed herein, SECX protein sequence of SEQ ID NOs:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 75, 77, 79 and 81, plus 30 and 32,or derivatives, fragments, analogs or homologs thereof, may be utilizedas immunogens in the generation of antibodies thatimmunospecifically-bind these protein components. The term “antibody” asused herein refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that specifically binds (immunoreactswith) an antigen, such as SECX. Such antibodies include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, F_(ab) andF_((ab′)2) fragments, and an F_(ab) expression library. In a specificembodiment, antibodies to human SECX proteins are disclosed. Variousprocedures known within the art may be used for the production ofpolyclonal or monoclonal antibodies to a SECX protein sequence, orderivative, fragment, analog or homolog thereof. Some of these proteinsare discussed below.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of the foregoing. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed SECX protein or achemically synthesized SECX polypeptide. The preparation can furtherinclude an adjuvant. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), humanadjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, orsimilar immunostimulatory agents. If desired, the antibody moleculesdirected against SECX can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction.

The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of SECX. A monoclonal antibody compositionthus typically displays a single binding affinity for a particular SECXprotein with which it immunoreacts. For preparation of monoclonalantibodies directed towards a particular SECX protein, or derivatives,fragments, analogs or homologs thereof, any technique that provides forthe production of antibody molecules by continuous cell line culture maybe utilized. Such techniques include, but are not limited to, thehybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497);the trioma technique; the human B-cell hybridoma technique (see Kozbor,et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique toproduce human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (seeCote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96). Each of the above citations are incorporated herein byreference in their entirety.

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a SECX protein (see e.g., U.S.Pat. No. 4,946,778). In addition, methodologies can be adapted for theconstruction of F_(ab) expression libraries (see e.g., Huse, et al.,1989 Science 246: 1275-1281) to allow rapid and effective identificationof monoclonal F_(ab) fragments with the desired specificity for a SECXprotein or derivatives, fragments, analogs or homologs thereof.Non-human antibodies can be “humanized” by techniques well known in theart. See e.g., U.S. Pat. No. 5,225,539. Antibody fragments that containthe idiotypes to a SECX protein may be produced by techniques known inthe art including, but not limited to: (i) an F_((ab′)2) fragmentproduced by pepsin digestion of an antibody molecule; (ii) an F_(ab)fragment generated by reducing the disulfide bridges of an F_((ab′)2)fragment; (iii) an F_(ab) fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) F_(v)fragments.

Additionally, recombinant anti-SECX antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in InternationalApplication No. PCT/US86/02269; European Patent Application No. 184,187;European Patent Application No. 171,496; European Patent Application No.173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No.4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No.125,023; Better et al.(1988) Science 240:1041-1043; Liu et al. (1987)PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526; Sun etal. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) JNatl Cancer Inst 80:1553-1559); Morrison(1985) Science 229:1202-1207; Oiet al. (1986) BioTechniques 4:214; Jones et al. (1986) Nature321:552-525; Verhoeyan et al. (1988) Scienc 239:1534; and Beidler et al.(1988) J Immunol 141:4053-4060. Each of the above citations areincorporated herein by reference in their entirety.

In one embodiment, methodologies for the screening of antibodies thatpossess the desired specificity include, but are not limited to,enzyme-linked immunosorbent assay (ELISA) and otherimmunologically-mediated techniques known within the art. In a specificembodiment, selection of antibodies that are specific to a particulardomain of a SECX protein is facilitated by generation of hybridomas thatbind to the fragment of a SECX protein possessing such a domain.Antibodies that are specific for an above-described domain within a SECXprotein, or derivatives, fragments, analogs or homologs thereof, arealso provided herein.

Anti-SECX antibodies may be used in methods known within the artrelating to the localization and/or quantitation of a SECX protein(e.g., for use in measuring levels of the SECX protein withinappropriate physiological samples, for use in diagnostic methods, foruse in imaging the protein, and the like). In a given embodiment,antibodies for SECX proteins, or derivatives, fragments, analogs orhomologs thereof, that contain the antibody derived binding domain, areutilized as pharmacologically-active compounds [hereinafter“Therapeutics”].

An anti-SECX antibody (e.g., monoclonal antibody) can be used to isolateSECX by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-SECX antibody can facilitate thepurification of natural SECX from cells and of recombinantly producedSECX expressed in host cells. Moreover, an anti-SECX antibody can beused to detect SECX protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the SECX protein. Anti-SECX antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

SECX Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding SECX protein, orderivatives, fragments, analogs or homologs thereof. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., SECX proteins, mutant forms ofSECX, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of SECX in prokaryotic or eukaryotic cells. For example, SECXcan be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the SECX expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) EMBO J 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp., San Diego, Calif.).

Alternatively, SECX can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include is the pAcseries (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g. milk whey promoter; U.S. Pat.No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to SECX mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosen thatdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen that direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al., “Antisense RNA asa molecular tool for genetic analysis,” Reviews—Trends in Genetics, Vol.1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, SECXprotein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding SECX or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) SECX protein.Accordingly, the invention further provides methods for producing SECXprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding SECX has been introduced) in asuitable medium such that SECX protein is produced. In anotherembodiment, the method further comprises isolating SECX from the mediumor the host cell.

Transgenic Animals

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichSECX-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous SECXsequences have been introduced into their genome or homologousrecombinant animals in which endogenous SECX sequences have beenaltered. Such animals are useful for studying the function and/oractivity of SECX and for identifying and/or evaluating modulators ofSECX activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA that is integrated into the genome of a cellfrom which a transgenic animal develops and that remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous SECX gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingSECX-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanSECX cDNA can be introduced as a transgene into the genome of anon-human animal. Alternatively, a nonhuman homologue of the human SECXgene, such as a mouse SECX gene, can be isolated based on hybridizationto the human SECX cDNA (described further above) and used as atransgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to the SECX transgene to direct expression of SECX protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan 1986, In:MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. Similar methods are used for production of othertransgenic animals. A transgenic founder animal can be identified basedupon the presence of the SECX transgene in its genome and/or expressionof SECX mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodingSECX can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a SECX gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the SECX gene. The SECX gene can be a human gene(e.g., the cDNA of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 74, 76, 78 and 80, plus 29 and 31), but more preferably, is anon-human homologue of a human SECX gene. For example, a mouse homologueof human SECX gene of, e.g., SEQ ID NO:29, can be used to construct ahomologous recombination vector suitable for altering an endogenous SECXgene in the mouse genome. In one embodiment, the vector is designed suchthat, upon homologous recombination, the endogenous SECX gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous SECX gene is mutated or otherwise alteredbut still encodes functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenousSECX protein). In the homologous recombination vector, the alteredportion of the SECX gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the SECX gene to allow for homologous recombination tooccur between the exogenous SECX gene carried by the vector and anendogenous SECX gene in an embryonic stem cell. The additional flankingSECX nucleic acid is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector.See e.g., Thomas et al. (1987) Cell 51:503 for a description ofhomologous recombination vectors. The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced SECX gene has homologously recombined with the endogenousSECX gene are selected (see e.g., Li et al. (1992) Cell 69:915).

The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. See e.g., Bradley 1987,In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH,Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley (1991) Curr Opin Biotechnol 2:823-829; PCTInternational Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968;and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedthat contain selected systems that allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.Another example of a recombinase system is the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.If a cre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Pharmaceutical Compositions

The SECX nucleic acid molecules, SECX proteins, and anti-SECX antibodies(also referred to herein as “active compounds”) of the invention, andderivatives, fragments, analogs and homologs thereof, can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a SECX protein or anti-SECX antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein that include extracellular and transmembrane domainsand, therefore, can be used in one or more of the following methods: (a)screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology), (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). A SECX protein interacting with other cellular proteinscan thus be used to (i) modulate that respective protein activity; (ii)regulate cellular proliferation; (iii) regulate cellulardifferentiation; and (iv) regulate cell survival.

The isolated nucleic acid molecules of the invention can be used toexpress SECX protein (e.g., via a recombinant expression vector in ahost cell in gene therapy applications), to detect SECX mRNA (e.g., in abiological sample) or a genetic lesion in a SECX gene, and to modulateSECX activity, as described further below. In addition, the SECXproteins can be used to screen drugs or compounds that modulate the SECXactivity or expression as well as to treat disorders characterized byinsufficient or excessive production of SECX protein or production ofSECX protein forms that have decreased or aberrant activity compared toSECX wild type protein (e.g. proliferative disorders such as cancer orpreclampsia, or any disease or disorder described in Sections 1-14above). In addition, the anti-SECX antibodies of the invention can beused to detect and isolate SECX proteins and modulate SECX activity.

This invention further pertains to novel agents identified by the abovedescribed screening assays and uses thereof for treatments as describedherein.

Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)that bind to SECX proteins or have a stimulatory or inhibitory effecton, for example, SECX expression or SECX activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of themembrane-bound form of a SECX protein or polypeptide or biologicallyactive portion thereof. The test compounds of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the “one-beadone-compound” library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam (1997) Anticancer Drug Des 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc Natl AcadSci U.S.A. 90:6909; Erb et al. (1994) Proc Natl Acad Sci U.S.A.91:11422; Zuckermann et al. (1994) J Med Chem 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew Chem Int Ed Engl 33:2059;Carell et al. (1994) Angew Chem Int Ed Engl 33:2061; and Gallop et al.(1994) J Med Chem 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) BioTechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), on chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc Natl Acad Sci U.S.A.87:6378-6382; Felici (1991) J Mol Biol 222:301-310; Ladner above.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of SECX protein, or a biologicallyactive portion thereof, on the cell surface is contacted with a testcompound and the ability of the test compound to bind to a SECX proteindetermined. The cell, for example, can of mammalian origin or a yeastcell. Determining the ability of the test compound to bind to the SECXprotein can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the SECX protein or biologically active portion thereof canbe determined by detecting the labeled compound in a complex. Forexample, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemission or by scintillation counting. Alternatively,test compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product. In one embodiment, the assay comprisescontacting a cell which expresses a membrane-bound form of SECX protein,or a biologically active portion thereof, on the cell surface with aknown compound which binds SECX to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a SECX protein, wherein determining theability of the test compound to interact with a SECX protein comprisesdetermining the ability of the test compound to preferentially bind toSECX or a biologically active portion thereof as compared to the knowncompound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of SECX protein, or abiologically active portion thereof, on the cell surface with a testcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the SECX protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of SECX or a biologically activeportion thereof can be accomplished, for example, by determining theability of the SECX protein to bind to or interact with a SECX targetmolecule. As used herein, a “target molecule” is a molecule with which aSECX protein binds or interacts in nature, for example, a molecule onthe surface of a cell which expresses a SECX interacting protein, amolecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. A SECX target molecule can bea non-SECX molecule or a SECX protein or polypeptide of the presentinvention. In one embodiment, a SECX target molecule is a component of asignal transduction pathway that facilitates transduction of anextracellular signal (e.g. a signal generated by binding of a compoundto a membrane-bound SECX molecule) through the cell membrane and intothe cell. The target, for example, can be a second intercellular proteinthat has catalytic activity or a protein that facilitates theassociation of downstream signaling molecules with SECX.

Determining the ability of the SECX protein to bind to or interact witha SECX target molecule can be accomplished by one of the methodsdescribed above for determining direct binding. In one embodiment,determining the ability of the SECX protein to bind to or interact witha SECX target molecule can be accomplished by determining the activityof the target molecule. For example, the activity of the target moleculecan be determined by detecting induction of a cellular second messengerof the target (i.e. intracellular Ca²⁺, diacylglycerol, IP₃, etc.),detecting catalytic/enzymatic activity of the target an appropriatesubstrate, detecting the induction of a reporter gene (comprising aSECX-responsive regulatory element operatively linked to a nucleic acidencoding a detectable marker, e.g., luciferase), or detecting a cellularresponse, for example, cell survival, cellular differentiation, or cellproliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a SECX protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the SECX protein or biologically activeportion thereof. Binding of the test compound to the SECX protein can bedetermined either directly or indirectly as described above. In oneembodiment, the assay comprises contacting the SECX protein orbiologically active portion thereof with a known compound which bindsSECX to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a SECX protein, wherein determining the ability of the testcompound to interact with a SECX protein comprises determining theability of the test compound to preferentially bind to SECX orbiologically active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting SECX protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g. stimulate or inhibit) the activity of the SECX protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of SECX can be accomplished, forexample, by determining the ability of the SECX protein to bind to aSECX target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of SECX can beaccomplished by determining the ability of the SECX protein furthermodulate a SECX target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theSECX protein or biologically active portion thereof with a knowncompound which binds SECX to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a SECX protein, wherein determining theability of the test compound to interact with a SECX protein comprisesdetermining the ability of the SECX protein to preferentially bind to ormodulate the activity of a SECX target molecule.

The cell-free assays of the present invention are amenable to use ofboth the soluble form or the membrane-bound form of SECX. In the case ofcell-free assays comprising the membrane-bound form of SECX, it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of SECX is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either SECX or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to SECX, or interaction of SECX with atarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided that adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, GST-SECX fusion proteins orGST-target fusion proteins can be adsorbed onto glutathione sepharosebeads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatizedmicrotiter plates, that are then combined with the test compound or thetest compound and either the non-adsorbed target protein or SECXprotein, and the mixture is incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads or microtiter plate wells are washed toremove any unbound components, the matrix immobilized in the case ofbeads, complex determined either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of SECX binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either SECX orits target molecule can be immobilized utilizing conjugation of biotinand streptavidin. Biotinylated SECX or target molecules can be preparedfrom biotin-NHS (N-hydroxy-succinimide) using techniques well known inthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with SECX or targetmolecules, but which do not interfere with binding of the SECX proteinto its target molecule, can be derivatized to the wells of the plate,and unbound target or SECX trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the SECX or target molecule, aswell as enzyme-linked assays that rely on detecting an enzymaticactivity associated with the SECX or target molecule.

In another embodiment, modulators of SECX expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of SECX mRNA or protein in the cell is determined. The levelof expression of SECX mRNA or protein in the presence of the candidatecompound is compared to the level of expression of SECX mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of SECX expression based on thiscomparison. For example, when expression of SECX mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of SECX mRNA or protein expression.Alternatively, when expression of SECX mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of SECX mRNA or protein expression. The level of SECX mRNA orprotein expression in the cells can be determined by methods describedherein for detecting SECX mRNA or protein.

In yet another aspect of the invention, the SECX proteins can be used as“bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)BioTechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins that bind to orinteract with SECX (“SECX-binding proteins” or “SECX-bp”) and modulateSECX activity. Such SECX-binding proteins are also likely to be involvedin the propagation of signals by the SECX proteins as, for example,upstream or downstream elements of the SECX pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for SECX is fused to agene encoding the DNA binding domain of a known transcription factor(e.g., GALA). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a SECX-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) that is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genethat encodes the protein which interacts with SECX.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the SECX, sequences, described herein, can beused to map the location of the SECX genes, respectively, on achromosome. The mapping of the SECX sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, SECX genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the SECX sequences.Computer analysis of the SECX, sequences can be used to rapidly selectprimers that do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the SECX sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes. (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the SECXsequences to design oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases, willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., HUMAN CHROMOSOMES: A MANUAL OFBASIC TECHNIQUES (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in McKusick,MENDELIAN INHERITANCE IN MAN, available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, for example, Egeland et al. (1987) Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the SECX gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

Tissue Typing

The SECX sequences of the present invention can also be used to identifyindividuals from minute biological samples. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentification. The sequences of the present invention are useful asadditional DNA markers for RFLP (“restriction fragment lengthpolymorphisms,” described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique that determines the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, theSECX sequences described herein can be used to prepare two PCR primersfrom the 5′ and 3′ ends of the sequences. These primers can then be usedto amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The SECX sequences of the invention uniquely represent portions of thehuman genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs),which include restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can be used as a standard againstwhich DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of any one or more of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 74, 76, 78 and 80, plus29 and 31 can comfortably provide positive individual identificationwith a panel of perhaps 10 to 1,000 primers that each yield a noncodingamplified sequence of 100 bases. If predicted coding sequences are used,a more appropriate number of primers for positive individualidentification would be 500-2,000.

A further use of the SECX sequences is to identify a cell or tissue typein a biological sample. As discussed above, various SECX genes areexpressed in one or more cell types. Thus, a cell type can be identifiedbased on the presence of RNA molecules from one or more SECX genes.Tissue distribution of various SECX genes are shown and discussed inFIGS. 19-23 and Examples 6-11, below.

Use of SECX Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, that can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e. another DNA sequence that is unique to a particularindividual). As mentioned above, actual base sequence information can beused for identification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SECX gene are particularly appropriate for this use, asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the SECX sequences or portions thereof,e.g., fragments derived from the noncoding regions of a SECX genedescribed herein, having a length of at least 20 bases, preferably atleast 30 bases.

The SECX sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes that can beused, for example, in an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue, etc. This can be very useful incases where a forensic pathologist is presented with a tissue of unknownorigin. Panels of such SECX probes can be used to identify tissue byspecies and/or by organ type.

In a similar fashion, these reagents, e.g., SECX primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

Determination of the Biological Effect of the Therapeutic

In various embodiments of the present invention, suitable in vitro or invivo assays are utilized to determine the effect of a specificTherapeutic and whether its administration is indicated for treatment ofthe affected tissue.

In various specific embodiments, in vitro assays may be performed withrepresentative cells of the type(s) involved in the patient's disorder,to determine if a given Therapeutic exerts the desired effect upon thecell type(s). Compounds for use in therapy may be tested in suitableanimal model systems including, but not limited to rats, mice, chicken,cows, monkeys, rabbits, and the like, prior to testing in humansubjects. Similarly, for in vivo testing, any of the animal model systemknown in the art may be used prior to administration to human subjects.

Malignancies

SECX proteins are located at the cellular membrane and are thought to beinvolved in the regulation of cell proliferation and differentiation.Accordingly, Therapeutics of the present invention may be useful in thetherapeutic or prophylactic treatment of diseases or disorders that areassociated with cell hyperproliferation and/or loss of control of cellproliferation (e.g., cancers, malignancies and tumors). For a review ofsuch hyperproliferation disorders, see e.g., Fishman, et al., 1985.MEDICINE, 2nd ed., J.B. Lippincott Co., Philadelphia, Pa.

Therapeutics of the present invention may be assayed by any method knownwithin the art for efficacy in treating or preventing malignancies andrelated disorders. Such assays include, but are not limited to, in vitroassays utilizing transformed cells or cells derived from the patient'stumor, as well as in vivo assays using animal models of cancer ormalignancies. Potentially effective Therapeutics are those that, forexample, inhibit the proliferation of tumor-derived or transformed cellsin culture or cause a regression of tumors in animal models, incomparison to the controls.

In the practice of the present invention, once a malignancy or cancerhas been shown to be amenable to treatment by modulating (i.e.,inhibiting, antagonizing or agonizing) activity, that cancer ormalignancy may subsequently be treated or prevented by theadministration of a Therapeutic that serves to modulate proteinfunction.

Premalignant Conditions

The Therapeutics of the present invention that are effective in thetherapeutic or prophylactic treatment of cancer or malignancies may alsobe administered for the treatment of pre-malignant conditions and/or toprevent the progression of a pre-malignancy to a neoplastic or malignantstate. Such prophylactic or therapeutic use is indicated in conditionsknown or suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia or, most particularly, dysplasia has occurred. For a reviewof such abnormal cell growth see e.g., Robbins & Angell, 1976. BASICPATHOLOGY, 2nd ed., W.B. Saunders Co., Philadelphia, Pa.

Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in its structure or function. For example, it has beendemonstrated that endometrial hyperplasia often precedes endometrialcancer. Metaplasia is a form of controlled cell growth in which one typeof mature or fully differentiated cell substitutes for another type ofmature cell. Metaplasia may occur in epithelial or connective tissuecells. Dysplasia is generally considered a precursor of cancer, and isfound mainly in the epithelia. Dysplasia is the most disorderly form ofnon-neoplastic cell growth, and involves a loss in individual celluniformity and in the architectural orientation of cells. Dysplasiacharacteristically occurs where there exists chronic irritation orinflammation, and is often found in the cervix, respiratory passages,oral cavity, and gall bladder.

Alternatively, or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed or malignant phenotypedisplayed either in vivo or in vitro within a cell sample derived from apatient, is indicative of the desirability of prophylactic/therapeuticadministration of a Therapeutic that possesses the ability to modulateactivity of An aforementioned protein. Characteristics of a transformedphenotype include, but are not limited to: (i) morphological changes;(ii) looser substratum attachment; (iii) loss of cell-to-cell contactinhibition; (iv) loss of anchorage dependence; (v) protease release;(vi) increased sugar transport; (vii) decreased serum requirement;(viii) expression of fetal antigens, (ix) disappearance of the 250 kDalcell-surface protein, and the like. See e.g., Richards, et al., 1986.MOLECULAR PATHOLOGY, W.B. Saunders Co., Philadelphia, Pa.

In a specific embodiment of the present invention, a patient thatexhibits one or more of the following predisposing factors formalignancy is treated by administration of an effective amount of aTherapeutic: (i) a chromosomal translocation associated with amalignancy (e.g., the Philadelphia chromosome (bcr/abl) for chronicmyelogenous leukemia and t(14;18) for follicular lymphoma, etc.); (ii)familial polyposis or Gardner's syndrome (possible forerunners of coloncancer); (iii) monoclonal gammopathy of undetermined significance (apossible precursor of multiple myeloma) and (iv) a first degree kinshipwith persons having a cancer or pre-cancerous disease showing aMendelian (genetic) inheritance pattern (e.g., familial polyposis of thecolon, Gardner's syndrome, hereditary exostosis, polyendocrineadenomatosis, Peutz-Jeghers syndrome, neurofibromatosis of VonRecklinghausen, medullary thyroid carcinoma with amyloid production andpheochromocytoma, retinoblastoma, carotid body tumor, cutaneousmelanocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum,ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi'saplastic anemia and Bloom's syndrome).

In another embodiment, a Therapeutic of the present invention isadministered to a human patient to prevent the progression to breast,colon, lung, pancreatic, or uterine cancer, or melanoma or sarcoma.

Hyperproliferative and Dysproliferative Disorders

In one embodiment of the present invention, a Therapeutic isadministered in the therapeutic or prophylactic treatment ofhyperproliferative or benign dysproliferative disorders. The efficacy intreating or preventing hyperproliferative diseases or disorders of aTherapeutic of the present invention may be assayed by any method knownwithin the art. Such assays include in vitro cell proliferation assays,in vitro or in vivo assays using animal models of hyperproliferativediseases or disorders, or the like. Potentially effective Therapeuticsmay, for example, promote cell proliferation in culture or cause growthor cell proliferation in animal models in comparison to controls.

Specific embodiments of the present invention are directed to thetreatment or prevention of cirrhosis of the liver (a condition in whichscarring has overtaken normal liver regeneration processes); treatmentof keloid (hypertrophic scar) formation causing disfiguring of the skinin which the scarring process interferes with normal renewal; psoriasis(a common skin condition characterized by excessive proliferation of theskin and delay in proper cell fate determination); benign tumors;fibrocystic conditions and tissue hypertrophy (e.g., benign prostatichypertrophy).

Neurodegenerative Disorders

SECX protein have been implicated in the deregulation of cellularmaturation and apoptosis, which are both characteristic ofneurodegenerative disease. Accordingly, Therapeutics of the invention,particularly but not limited to those that modulate (or supply) activityof an aforementioned protein, may be effective in treating or preventingneurodegenerative disease. Therapeutics of the present invention thatmodulate the activity of an aforementioned protein involved inneurodegenerative disorders can be assayed by any method known in theart for efficacy in treating or preventing such neurodegenerativediseases and disorders. Such assays include in vitro assays forregulated cell maturation or inhibition of apoptosis or in vivo assaysusing animal models of neurodegenerative diseases or disorders, or anyof the assays described below. Potentially effective Therapeutics, forexample but not by way of limitation, promote regulated cell maturationand prevent cell apoptosis in culture, or reduce neurodegeneration inanimal models in comparison to controls.

Once a neurodegenerative disease or disorder has been shown to beamenable to treatment by modulation activity, that neurodegenerativedisease or disorder can be treated or prevented by administration of aTherapeutic that modulates activity. Such diseases include alldegenerative disorders involved with aging, especially osteoarthritisand neurodegenerative disorders.

Disorders Related to Organ Transplantation

SECX has been implicated in disorders related to organ transplantation,in particular but not limited to organ rejection. Therapeutics of theinvention, particularly those that modulate (or supply) activity, may beeffective in treating or preventing diseases or disorders related toorgan transplantation. Therapeutics of the invention (particularlyTherapeutics that modulate the levels or activity of an aforementionedprotein) can be assayed by any method known in the art for efficacy intreating or preventing such diseases and disorders related to organtransplantation. Such assays include in vitro assats for using cellculture models as described below, or in vivo assays using animal modelsof diseases and disorders related to organ transplantation, see e.g.below. Potentially effective Therapeutics, for example but not by way oflimitation, reduce immune rejection responses in animal models incomparison to controls.

Accordingly, once diseases and disorders related to organtransplantation are shown to be amenable to treatment by modulation ofactivity, such diseases or disorders can be treated or prevented byadministration of a Therapeutic that modulates activity.

Cardiovascular Disease

SECX has been implicated in cardiovascular disorders, including inatherosclerotic plaque formation. Diseases such as cardiovasculardisease, including cerebral thrombosis or hemorrhage, ischemic heart orrenal disease, peripheral vascular disease, or thrombosis of other majorvessel, and other diseases, including diabetes mellitus, hypertension,hypothyroidism, cholesterol ester storage disease, systemic lupuserythematosus, homocysteinemia, and familial protein or lipid processingdiseases, and the like, are either directly or indirectly associatedwith atherosclerosis. Accordingly, Therapeutics of the invention,particularly those that modulate (or supply) activity or formation maybe effective in treating or preventing atherosclerosis-associateddiseases or disorders. Therapeutics of the invention (particularlyTherapeutics that modulate the levels or activity) can be assayed by anymethod known in the art, including those described below, for efficacyin treating or preventing such diseases and disorders.

A vast array of animal and cell culture models exist for processesinvolved in atherosclerosis. A limited and non-exclusive list of animalmodels includes knockout mice for premature atherosclerosis (Kurabayashiand Yazaki, 1996, Int. Angiol. 15: 187-194), transgenic mouse models ofatherosclerosis (Kappel et al., 1994, FASEB J. 8: 583-592), antisenseoligonucleotide treatment of animal models (Callow, 1995, Curr. Opin.Cardiol. 10: 569-576), transgenic rabbit models for atherosclerosis(Taylor, 1997, Ann. N.Y. Acad. Sci 811: 146-152), hypercholesterolemicanimal models (Rosenfeld, 1996, Diabetes Res. Clin. Pract. 30 Suppl.:1-11), hyperlipidemic mice (Paigen et al., 1994, Curr. Opin. Lipidol. 5:258-264), and inhibition of lipoxygenase in animals (Sigal et al., 1994,Ann. N.Y. Acad. Sci. 714: 211-224). In addition, in vitro cell modelsinclude but are not limited to monocytes exposed to low densitylipoprotein (Frostegard et al., 1996, Atherosclerosis 121: 93-103),cloned vascular smooth muscle cells (Suttles et al., 1995, Exp. CellRes. 218: 331-338), endothelial cell-derived chemoattractant exposed Tcells (Katz et al., 1994, J. Leukoc. Biol. 55: 567-573), cultured humanaortic endothelial cells (Farber et al., 1992, Am. J. Physiol. 262:H1088-1085), and foam cell cultures (Libby et al., 1996, Curr OpinLipidol 7: 330-335). Potentially effective Therapeutics, for example butnot by way of limitation, reduce foam cell formation in cell culturemodels, or reduce atherosclerotic plaque formation inhypercholesterolemic mouse models of atherosclerosis in comparison tocontrols.

Accordingly, once an atherosclerosis-associated disease or disorder hasbeen shown to be amenable to treatment by modulation of activity orformation, that disease or disorder can be treated or prevented byadministration of a Therapeutic that modulates activity.

Cytokine and Cell Proliferation/Differentiation Activity

A SECX protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations. Many protein factorsdiscovered to date, including all known cytokines, have exhibitedactivity in one or more factor dependent cell proliferation assays, andhence the assays serve as a convenient confirmation of cytokineactivity. The activity of a protein of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines including, without limitation, 32D,DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.

The activity of a protein of the invention may, among other means, bemeasured by the following methods: Assays for T-cell or thymocyteproliferation include without limitation those described in: CURRENTPROTOCOLS IN IMMUNOLOGY, Ed by Coligan et al., Greene PublishingAssociates and Wiley-Interscience (Chapter 3 and Chapter 7); Takai etal., J Immunol 137:3494-3500, 1986; Bertagnoili et al., J Immunol145:1706-1712, 1990; Bertagnolli et al., Cell Immunol 133:327-341, 1991;Bertagnolli, et al., J Immunol 149:3778-3783, 1992; Bowman et al., JImmunol 152:1756-1761, 1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed by Kruisbeek and Shevach, In: CURRENT PROTOCOLS IN IMMUNOLOGY.Coligan et al., eds. Vol 1, pp. 3.12.1-14, John Wiley and Sons, Toronto1994; and by Schreiber, In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coliganeds. Vol 1 pp. 6.8.1-8, John Wiley and Sons, Toronto 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described byBottomly et al., In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al.,eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto 1991; deVrieset al., J Exp Med 173:1205-1211, 1991; Moreau et al., Nature336:690-692, 1988; Greenberger et al., Proc Natl Acad Sci U.S.A.80:2931-2938, 1983; Nordan, In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coliganet al., eds. Vol 1 pp. 6.6.1-5, John Wiley and Sons, Toronto 1991; Smithet al., Proc Natl Acad Sci U.S.A. 83:1857-1861, 1986; Measurement ofhuman Interleukin 11-Bennett, et al. In: CURRENT PROTOCOLS INIMMUNOLOGY. Coligan et al., eds. Vol 1 pp. 6.15.1 John Wiley and Sons,Toronto 1991; Ciarletta, et al., In: CURRENT PROTOCOLS IN IMMUNOLOGY.Coligan et al., eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described In: CURRENTPROTOCOLS IN IMMUNOLOGY. Coligan et al., eds., Greene PublishingAssociates and Wiley-Interscience (Chapter 3Chapter 6, Chapter 7);Weinberger et al., Proc Natl Acad Sci USA 77:6091-6095, 1980; Weinbergeret al., Eur J Immun 11:405-411, 1981; Takai et al., J Immunol137:3494-3500, 1986; Takai et al., J Immunol 140:508-512, 1988.

Immune Stimulating or Suppressing Activity

A SECX protein of the present invention may also exhibit immunestimulating or immune suppressing activity, including without limitationthe activities for which assays are described herein. A protein may beuseful in the treatment of various immune deficiencies and disorders(including severe combined immunodeficiency (SCID)), e.g., in regulating(up or down) growth and proliferation of T and/or B lymphocytes, as wellas effecting the cytolytic activity of NK cells and other cellpopulations. These immune deficiencies may be genetic or be caused byvital (e.g., HIV) as well as bacterial or fungal infections, or mayresult from autoimmune disorders. More specifically, infectious diseasescauses by vital, bacterial, fungal or other infection may be treatableusing a protein of the present invention, including infections by HIV,hepatitis viruses, herpesviruses, mycobacteria, Leishmania species.,malaria species, and various fungal infections such as candidiasis. Ofcourse, in this regard, a protein of the present invention may also beuseful where a boost to the immune system generally may be desirable,i.e., in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitus, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein of the present invention may also to be useful in thetreatment of allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems. Otherconditions, in which immune suppression is desired (including, forexample, organ transplantation), may also be treatable using a proteinof the present invention.

Using the proteins of the invention it may also be possible to immuneresponses, in a number of ways. Down regulation may be in the form ofinhibiting or blocking an immune response already in progress or mayinvolve preventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or energyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon re-exposure to specific antigen in theabsence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this matter prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to energize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of B lymphocyte antigens.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc Natl Acad Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., FUNDAMENTAL IMMUNOLOGY,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking B lymphocyte antigen function in vivo on thedevelopment of that disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and auto-antibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of T cells bydisrupting receptor:ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofauto-antibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythematosis in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,FUNDAMENTAL IMMUNOLOGY, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function may be useful in cases of viral infection.In addition, systemic vital diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-vital immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

In another application, up regulation or enhancement of antigen function(preferably B lymphocyte antigen function) may be useful in theinduction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleicacid encoding at least one peptide of the present invention can beadministered to a subject to overcome tumor-specific tolerance in thesubject. If desired, the tumor cell can be transfected to express acombination of peptides. For example, tumor cells obtained from apatient can be transfected ex vivo with an expression vector directingthe expression of a peptide having B7-2-like activity alone, or inconjunction with a peptide having B7-1-like activity and/or B7-3-likeactivity. The transfected tumor cells are returned to the patient toresult in expression of the peptides on the surface of the transfectedcell. Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activityof a B lymphocyte antigen(s) on the surface of the tumor cell providesthe necessary costimulation signal to T cells to induce a T cellmediated immune response against the transfected tumor cells. Inaddition, tumor cells which lack MHC class I or MHC class II molecules,or which fail to reexpress sufficient amounts of MHC class I or MHCclass II molecules, can be transfected with nucleic acid encoding all ora portion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I αchain protein and β₂ microglobulin protein or an MHC class II achain protein and an MHC class II β chain protein to thereby express MHCclass I or MHC class II proteins on the cell surface. Expression of theappropriate class I or class II MHC in conjunction with a peptide havingthe activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) inducesa T cell mediated immune response against the transfected tumor cell.Optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, bemeasured by the following methods: Suitable assays for thymocyte orsplenocyte cytotoxicity include, without limitation, those described In:CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al., eds. Greene PublishingAssociates and Wiley-Interscience (Chapter 3, Chapter 7); Herrmann etal., Proc Natl Acad Sci USA 78:2488-2492, 1981; Herrmann et a., JImmunol 128:1968-1974, 1982; Handa et al., J Immunol 135:1564-1572,1985; Takai et al., J Immunol 137:3494-3500, 1986; Takai et al., JImmunol 140:508-512, 1988; Herrmann et al., Proc Natl Acad Sci USA78:2488-2492, 1981; Herrmann et al., J Immunol 128:1968-1974, 1982;Handa et al., J Immunol 135:1564-1572,1985; Takai et al., J Immunol137:3494-3500,1986; Bowman et al., J Virology 61:1992-1998; Takai et al,J Immunol 140:508-512, 1988; Bertagnolli et al., Cell Immunol133:327-341, 1991; Brown et al., J Immunol 153:3079-3092, 1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J Immunol144:3028-3033, 1990; and Mond and Brunswick In: CURRENT PROTOCOLS INIMMUNOLOGY. Coligan et al., (eds.) Vol 1 pp. 3.8.1-3.8.16, John Wileyand Sons, Toronto 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described In: CURRENT PROTOCOLS INIMMUNOLOGY. Coligan et al., eds. Greene Publishing Associates andWiley-Interscience (Chapter 3, Chapter 7); Takai et al., J Immunol137:3494-3500, 1986; Takai et al., J Immunol 140:508-512, 1988;Bertagnolli et al., J Immunol 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J Immunol134:536-544, 1995; Inaba et al., J Exp Med 173:549-559, 1991; Macatoniaet al., J Immunol 154:5071-5079, 1995; Porgador et al., J Exp Med182:255-260, 1995; Nair et al., J Virol 67:4062-4069, 1993; Huang etal., Science 264:961-965, 1994; Macatonia et al, J Exp Med169:1255-1264, 1989; Bhardwaj et al., J Clin Investig 94:797-807, 1994;and Inaba et al., J Exp Med 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Res 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;Zacharchuk, J Immunol 145:4037-4045, 1990; Zamai et al., Cytometry14:891-897,1993; Gorczyca et al., Internat J Oncol 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cell Immunol 155: 111-122,1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc Nat AcadSci USA 88:7548-7551, 1991.

Hematopoiesis Regulating Activity

A SECX protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Mol.Cell. Biol. 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915,1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, In: CULTURE OF HEMATOPOIETIC CELLS. Freshney, et al.(eds.) Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayamaet al., Proc Natl Acad Sci USA 89:5907-5911, 1992; McNiece and Briddeli,In: CULTURE OF HEMATOPOIETIC CELLS. Freshney, et al. (eds.) Vol pp.23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Exp Hematol22:353-359, 1994; Ploemacher, In: CULTURE OF HEMATOPOIETIC CELLS.Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y.1994; Spooncer et al., In: CULTURE OF HEMATOPOIETIC CELLS. Freshhey, etal., (eds.) Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994;Sutherland, In: CULTURE OF HEMATOPOIETIC CELLS. Freshney, et al., (eds.)Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

Tissue Growth Activity

A SECX protein of the present invention also may have utility incompositions used for bone, cartilage, tendon, ligament and/or nervetissue growth or regeneration, as well as for wound healing and tissuerepair and replacement, and in the treatment of bums, incisions andulcers.

A protein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein of this invention may also be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein of the invention may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes.

Another category of tissue regeneration activity that may beattributable to the protein of the present invention is tendon/ligamentformation. A protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide an environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce groeth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendonitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a career as is wellknown in the art.

The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a protein may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or fasterclosure of non-healing wounds, including without limitation pressureulcers, ulcers associated with vascular insufficiency, surgical andtraumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibitactivity for generation or regeneration of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac) and vascular(including vascular endothelium) tissue, or for promoting the growth ofcells comprising such tissues. Part of the desired effects may be byinhibition or modulation of fibrotic scarring to allow normal tissue toregenerate. A protein of the invention may also exhibit angiogenicactivity.

A protein of the present invention may also be useful for gut protectionor regeneration and treatment of lung or liver fibrosis, reperfusioninjury in various tissues, and conditions resulting from systemiccytokine damage.

A protein of the present invention may also be useful for promoting orinhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, EPIDERMAL WOUND HEALING, pp. 71-112 (Maibach andRovee, eds.), Year Book Medical Publishers, Inc., Chicago, as modifiedby Eaglstein and Menz, J. Invest. Dermatol 71:382-84 (1978).

Activin/Inhibin Activity

A SECX protein of the present invention may also exhibit activin- orinhibin-related activities. Inhibins are characterized by their abilityto inhibit the release of follicle stimulating hormone (FSH), whileactivins and are characterized by their ability to stimulate the releaseof follicle stimulating hormone (FSH). Thus, a protein of the presentinvention, alone or in heterodimers with a member of the inhibin afamily, may be useful as a contraceptive based on the ability ofinhibins to decrease fertility in female mammals and decreasespermatogenesis in male mammals. Administration of sufficient amounts ofother inhibins can induce infertility in these mammals. Alternatively,the protein of the invention, as a homodimer or as a heterodimer withother protein subunits of the inhibin-b group, may be useful as afertility inducing therapeutic, based upon the ability of activinmolecules in stimulating FSH release from cells of the anteriorpituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of theinvention may also be useful for advancement of the onset of fertilityin sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al.,Nature 321:779-782, 1986: Vale et al., Nature 321:776-779, 1986; Masonet al, Nature 318:659-663, 1985; Forage et al., Proc Natl Acad Sci USA83:3091-3095, 1986.

Chemotactic/Chemokinetic Activity

A protein of the present invention may have chemotactic or chemokineticactivity (e.g., act as a chemokine) for mammalian cells, including, forexample, monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells. Chemotactic andchemokinetic proteins can be used to mobilize or attract a desired cellpopulation to a desired site of action. Chemotactic or chemokineticproteins provide particular advantages in treatment of wounds and othertrauma to tissues, as well as in treatment of localized infections. Forexample, attraction of lymphocytes, monocytes or neutrophils to tumorsor sites of infection may result in improved immune responses againstthe tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

The activity of a protein of the invention may, among other means, bemeasured by following methods. Assays for chemotactic activity (whichwill identify proteins that induce or prevent chemotaxis) consist ofassays that measure the ability of a protein to induce the migration ofcells across a membrane as well as the ability of a protein to inducethe adhesion of one cell population to another cell population. Suitableassays for movement and adhesion include, without limitation, thosedescribed in: CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al., eds.(Chapter 6.12, MEASUREMENT OF ALPHA AND BETA CHEMOKINES 6.12.1-6.12.28);Taub et al. J Clin Invest 95:1370-1376, 1995; Lind et al. APMIS103:140-146, 1995; Muller et al., Eur J Immunol 25: 1744-1748; Gruberetal. J Immunol 152:5860-5867, 1994; Johnston et al., J Immunol 153:1762-1768, 1994.

Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolyticactivity. As a result, such a protein is expected to be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A protein of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

Receptor/Ligand Activity

A protein of the present invention may also demonstrate activity asreceptors, receptor ligands or inhibitors or agonists of receptor/ligandinteractions. Examples of such receptors and ligands include, withoutlimitation, cytokine receptors and their ligands, receptor kinases andtheir ligands, receptor phosphatases and their ligands, receptorsinvolved in cell—cell interactions and their ligands (including withoutlimitation, cellular adhesion molecules (such as selecting, integrinsand their ligands) and receptor/ligand pairs involved in antigenpresentation, antigen recognition and development of cellular andhumoral immune responses). Receptors and ligands are also useful forscreening of potential peptide or small molecule inhibitors of therelevant receptor/ligand interaction. A protein of the present invention(including, without limitation, fragments of receptors and ligands) maythemselves be useful as inhibitors of receptor/ligand interactions.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: CURRENT PROTOCOLS IN IMMUNOLOGY, Ed by Coligan, etal., Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc Natl Acad Sci USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J ImmunolMethods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

Anti-Inflammatory Activity

Proteins of the present invention may also exhibit anti-inflammatoryactivity. The anti-inflammatory activity may be achieved by providing astimulus to cells involved in the inflammatory response, by inhibitingor promoting cell—cell interactions (such as, for example, celladhesion), by inhibiting or promoting chemotaxis of cells involved inthe inflammatory process, inhibiting or promoting cell extravasation, orby stimulating or suppressing production of other factors which moredirectly inhibit or promote an inflammatory response. Proteinsexhibiting such activities can be used to treat inflammatory conditionsincluding chronic or acute conditions), including without limitationinflammation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins of the inventionmay also be useful to treat anaphylaxis and hypersensitivity to anantigenic substance or material.

Tumor Inhibition Activity

In addition to the activities described above for immunologicaltreatment or prevention of tumors, a protein of the invention mayexhibit other anti-tumor activities. A protein may inhibit tumor growthdirectly or indirectly (such as, for example, via ADCC). A protein mayexhibit its tumor inhibitory activity by acting on tumor tissue or tumorprecursor tissue, by inhibiting formation of tissues necessary tosupport tumor growth (such as, for example, by inhibiting angiogenesis),by causing production of other factors, agents or cell types whichinhibit tumor growth, or by suppressing, eliminating or inhibitingfactors, agents or cell types which promote tumor growth.

Other Activities

A protein of the invention may also exhibit one or more of the followingadditional activities or effects: inhibiting the growth, infection orfunction of, or killing, infectious agents, including, withoutlimitation, bacteria, viruses, fungi and other parasites; effecting(suppressing or enhancing) bodily characteristics, including, withoutlimitation, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution, change in boneform or shape); effecting biorhythms or circadian cycles or rhythms;effecting the fertility of male or female subjects; effecting themetabolism, catabolism, anabolism, processing, utilization, storage orelimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, cofactors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningSECX protein and/or nucleic acid expression as well as SECX activity, inthe context of a biological sample (e.g. blood, serum, cells, tissue) tothereby determine whether an individual is afflicted with a disease ordisorder, or is at risk of developing a disorder, associated withaberrant SECX expression or activity. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing a disorder associated with SECX protein,nucleic acid expression or activity. For example, mutations in a SECXgene can be assayed in a biological sample. Such assays can be used forprognostic or predictive purpose to thereby prophylactically treat anindividual prior to the onset of a disorder characterized by orassociated with SECX protein, nucleic acid expression or activity.

Another aspect of the invention provides methods for determining SECXprotein, nucleic acid expression or SECX activity in an individual tothereby select appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as “pharmacogenomics”). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs, compounds) on the expression or activity of SECXin clinical trials.

These and other agents are described in further detail in the followingsections.

Diagnostic Assays

An exemplary method for detecting the presence or absence of SECX in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting SECX protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes SECX protein such that the presence of SECX isdetected in the biological sample. An agent for detecting SECX mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toSECX mRNA or genomic DNA. The nucleic acid probe can be, for example, afull-length SECX nucleic acid, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to SECX mRNA or genomic DNA. Other suitable probes for use inthe diagnostic assays of the invention are described herein.

An agent for detecting SECX protein is an antibody capable of binding toSECX protein, preferably an antibody with a detectable label. Antibodiescan be polyclonal, or more preferably, monoclonal. An intact antibody,or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. The term“labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect SECX mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of SECX mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of SECX proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of SECX genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of SECX protein includeintroducing into a subject a labeled anti-SECX antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting SECX protein, mRNA, orgenomic DNA, such that the presence of SECX protein, mRNA or genomic DNAis detected in the biological sample, and comparing the presence of SECXprotein, mRNA or genomic DNA in the control sample with the presence ofSECX protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of SECXin a biological sample. For example, the kit can comprise: a labeledcompound or agent capable of detecting SECX protein or mRNA in abiological sample; means for determining the amount of SECX in thesample; and means for comparing the amount of SECX in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectSECX protein or nucleic acid.

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant SECX expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with SECX protein, nucleic acidexpression or activity such as cancer or fibrotic disorders, or aSECX-spesific disease as described in the individual sections 1-14,above. Alternatively, the prognostic assays can be utilized to identifya subject having or at risk for developing a disease or disorder. Thus,the present invention provides a method for identifying a disease ordisorder associated with aberrant SECX expression or activity in which atest sample is obtained from a subject and SECX protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of SECXprotein or nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant SECXexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant SECX expression or activity. For example, suchmethods can be used to determine whether a subject can be effectivelytreated with an agent for a disorder, such as cancer or preclampsia or aSECX-specific disease as described in the individual sections 1-14,above. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant SECX expression or activity in which atest sample is obtained and SECX protein or nucleic acid is detected(e.g., wherein the presence of SECX protein or nucleic acid isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant SECX expression or activity.)

The methods of the invention can also be used to detect genetic lesionsin a SECX gene, thereby determining if a subject with the lesioned geneis at risk for a disorder characterized by aberrant cell proliferationand/or differentiation. In various embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion characterized by at least one of analteration affecting the integrity of a gene encoding a SECX-protein, orthe mis-expression of the SECX gene. For example, such genetic lesionscan be detected by ascertaining the existence of at least one of (1) adeletion of one or more nucleotides from a SECX gene; (2) an addition ofone or more nucleotides to a SECX gene; (3) a substitution of one ormore nucleotides of a SECX gene, (4) a chromosomal rearrangement of aSECX gene; (5) an alteration in the level of a messenger RNA transcriptof a SECX gene, (6) aberrant modification of a SECX gene, such as of themethylation pattern of the genomic DNA, (7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a SECX gene, (8)a non-wild type level of a SECX-protein, (9) allelic loss of a SECXgene, and (10) inappropriate post-translational modification of aSECX-protein. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in aSECX gene. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject. However, anybiological sample containing nucleated cells may be used, including, forexample, buccal mucosal cells.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the SECX-ene (see Abravaya et al. (1995)Nucl Acids Res 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers that specificallyhybridize to a SECX gene under conditions such that hybridization andamplification of the SECX gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al., 1990, Proc Natl Acad Sci USA87:1874-1878), transcriptional amplification system (Kwoh, et al., 1989,Proc Natl Acad Sci USA 86:1173-1177), Q-Beta Replicase (Lizardi et al,1988, BioTechnology 6:1197), or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a SECX gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,493,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in SECX can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin et al. (1996) Human Mutation 7: 244-255; Kozal et al.(1996) Nature Medicine 2: 753-759). For example, genetic mutations inSECX can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin et al. above. Briefly,a first hybridization array of probes can be used to scan through longstretches of DNA in a sample and control to identify base changesbetween the sequences by making linear arrays of sequential overlappingprobes. This step allows the identification of point mutations. Thisstep is followed by a second hybridization array that allows thecharacterization of specific mutations by using smaller, specializedprobe arrays complementary to all variants or mutations detected. Eachmutation array is composed of parallel probe sets, one complementary tothe wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the SECX gene anddetect mutations by comparing the sequence of the sample SECX with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert (1977) PNAS 74:560 or Sanger (1977) PNAS 74:5463. It is alsocontemplated that any of a variety of automated sequencing procedurescan be utilized when performing the diagnostic assays (Naeve et al.,(1995) BioTechniques 19:448), including sequencing by mass spectrometry(see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al. (1996)Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147-159).

Other methods for detecting mutations in the SECX gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type SECX sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent that cleaves single-stranded regions of the duplexsuch as which will exist due to basepair mismatches between the controland sample strands. For instance, RNA/DNA duplexes can be treated withRNase and DNA/DNA hybrids treated with S1 nuclease to enzymaticallydigesting the mismatched regions. In other embodiments, either DNA/DNAor RNA/DNA duplexes can be treated with hydroxylamine or osmiumtetroxide and with piperidine in order to digest mismatched regions.After digestion of the mismatched regions, the resulting material isthen separated by size on denaturing polyacrylamide gels to determinethe site of mutation. See, for example, Cotton et al (1988) Proc NatlAcad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol 217:286-295.In an embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in SECX cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a SECX sequence,e.g., a wild-type SECX sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in SECX genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl Acad Sci USA: 86:2766, see also Cotton(1993) Mutat Res 285:125-144; Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control SECXnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In one embodiment,the subject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing gradient to identify differences in the mobility ofcontrol and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc Natl Acad Sci USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology that depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al (1992) Mol Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc Nll Acad Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence, making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a SECX gene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which SECX is expressed may be utilized in the prognosticassays described herein. However, any biological sample containingnucleated cells may be used, including, for example, buccal mucosalcells.

Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onSECX activity (e.g., SECX gene expression), as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) disorders (e.g., cancer orgestational disorders or a SECX-specific disease as described in theindividual sections 1-14, above) associated with aberrant SECX activity.In conjunction with such treatment, the pharmacogenomics (i.e., thestudy of the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of SECX protein,expression of SECX nucleic acid, or mutation content of SECX genes in anindividual can be determined to thereby select appropriate agent(s) fortherapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum, Clin Exp PharmacolPhysiol, 1996, 23:983-985 and Linder, Clin Chem, 1997, 43:254-266. Ingeneral, two types of pharmacogenetic conditions can be differentiated.Genetic conditions transmitted as a single factor altering the way drugsact on the body (altered drug action) or genetic conditions transmittedas single factors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raredefects or as polymorphisms. For example, glucose-6-phosphatedehydrogenase (G6PD) deficiency is a common inherited enzymopathy inwhich the main clinical complication is haemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of SECX protein, expression of SECX nucleic acid, ormutation content of SECX genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual. In addition, pharmacogenetic studies can beused to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a SECX modulator, such as a modulator identified by one of theexemplary screening assays described herein.

Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of SECX (e.g., the ability to modulate aberrantcell proliferation and/or differentiation) can be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase SECX gene expression, protein levels, or upregulateSECX activity, can be monitored in clinical trails of subjectsexhibiting decreased SECX gene expression, protein levels, ordownregulated SECX activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease SECX gene expression,protein levels, or downregulate SECX activity, can be monitored inclinical trails of subjects exhibiting increased SECX gene expression,protein levels, or upregulated SECX activity. In such clinical trials,the expression or activity of SECX and, preferably, other genes thathave been implicated in, for example, a cellular proliferation disorderor a SECX-specific disease as described in the individual sections 1-14,above, can be used as a “read out” or markers of the immuneresponsiveness of a particular cell.

For example, and not by way of limitation, genes, including SECX, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) that modulates SECX activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on cellular proliferation disorders, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of SECX and other genes implicated in thedisorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of SECX or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

In one embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a SECX protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the SECX protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the SECX protein, mRNA, or genomic DNA inthe pre-administration sample with the SECX protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of SECX to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of SECX to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant SECX expression oractivity.

Disorders

Diseases and disorders that are characterized by increased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that antagonize(i.e., reduce or inhibit) activity. Therapeutics that antagonizeactivity may be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, (i)an aforementioned peptide, or analogs, derivatives, fragments orhomologs thereof; (ii) antibodies to an aforementioned peptide; (iii)nucleic acids encoding an aforementioned peptide; (iv) administration ofantisense nucleic acid and nucleic acids that are “dysfunctional” (i.e.,due to a heterologous insertion within the coding sequences of codingsequences to an aforementioned peptide) that are utilized to “knockout”endogenous function of an aforementioned peptide by homologousrecombination (see, e.g., Capecchi, 1989, Science 244: 1288-1292); or(v) modulators (i.e., inhibitors, agonists and antagonists, includingadditional peptide mimetic of the invention or antibodies specific to apeptide of the invention) that alter the interaction between anaforementioned peptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that increase(i.e., are agonists to) activity. Therapeutics that upregulate activitymay be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, anaforementioned peptide, or analogs, derivatives, fragments or homologsthereof; or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of anaforementioned peptide). Methods that are well-known within the artinclude, but are not limited to, immunoassays (e.g. by Western blotanalysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, etc.).

Prophylactic Methods

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant SECXexpression or activity, by administering to the subject an agent thatmodulates SECX expression or at least one SECX activity. Subjects atrisk for a disease that is caused or contributed to by aberrant SECXexpression or activity can be identified by, for example, any or a iscombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the SECX aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of SECX aberrancy, for example, aSECX agonist or SECX antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein. The prophylactic methods of the presentinvention are further discussed in the following subsections.

Therapeutic Methods

Another aspect of the invention pertains to methods of modulating SECXexpression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of SECX protein activity associated withthe cell. An agent that modulates SECX protein activity can be an agentas described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a SECX protein, a peptide, a SECXpeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more SECX protein activity. Examples of suchstimulatory agents include active SECX protein and a nucleic acidmolecule encoding SECX that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more SECX proteinactivity. Examples of such inhibitory agents include antisense SECXnucleic acid molecules and anti-SECX antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a SECX protein or nucleic acidmolecule. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) SECX expression or activity. In another embodiment, themethod involves administering a SECX protein or nucleic acid molecule astherapy to compensate for reduced or aberrant SECX expression oractivity.

Stimulation of SECX activity is desirable in situations in which SECX isabnormally downregulated and/or in which increased SECX activity islikely to have a beneficial effect. One example of such a situation iswhere a subject has a disorder characterized by aberrant cellproliferation and/or differentiation (e.g., cancer). Another example ofsuch a situation is where the subject has a gestational disease (e.g.,preclampsia). Other diseases of the invention include the SECX-specificdiseases as described in the individual sections 1-14, above.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLES Example 1 Radiation Hybrid Mapping Identifies the ChromosomalLocation of the Clones of the Invention

Radiation hybrid mapping using human chromosome markers was carried outfor many of the clones described in the present invention. The procedureused to obtain these results is analogous to methods known in the art,for example, Steen, et al. 1999 Genome Research 9: AP1-AP8. A panel of93 cell clones containing randomized radiation-induced human chromosomalfragments was screened in 96 well plates using PCR primers designed toidentify the given clones in a unique fashion. The results are presentedin Table 2, which provides the clone number, the chromosome on which theclone is found, the distance in cR from a marker gene to the soughtclone, and the identity of the marker gene.

TABLE 2 Radiation Hybrid Mapping Results for Clones of the InventionChromosome Distance, Clone No. No. cR Marker Gene 2777610 3 564.40AFM320WD1 2864933-1 and 2864933-2 5 316.40 WI-9907 2982339 3 355.00AFM320WD1 3911675 10 391.30 IB3079 4004731-1 12 404.60 WI-5272 403550811 230.10 WI-4920 4339264 19 311.50 IB1264

Example 2 Molecular Cloning of 2864933-1

The predicted open reading frame for the 2864933-1 protein codes for a939 amino acid long Type I transmembrane protein with an overall 95%identity to the mouse semaphorin Via protein. The predicted signalpeptide sequence is between residues 1-18, and the predictedtransmembrane domain is between residues 645-661. A fragment of the cDNAfor the 2864933-1 protein, coding for the extracellular domain ofpredicted mature protein (i.e., after removal of the signal peptide)from residue 19 to 644, has been cloned from human fetal brain cDNA.

The following oligonucleotide primers were designed to amplify thesought mature form of 2864933-1 4 by PCR:

2864933 MatF

GGATCC GGT TTC CCA GAA GAT TCT GAG CCA ATC (SEQ ID NO:33)

2864933 F-TOPO-Reverse

CTC GAG CTG GTC GTG GCC TTT GAG GTA ACT TTC (SEQ ID NO:34)

For cloning purposes, the forward primer includes an in frame BamHIrestriction site and the reverse primer contains an in frame XhoIrestriction site.

PCR reactions were set up using 5 ng human fetal brain cDNA. Thereaction mixture contained 1 microM of each if the 2864933 MatF and2864933 F-TOPO-Reverse primers, 5 micromoles dNTP (ClontechLaboratories, Palo Alto Calif.) and 1 microliter of 50× Advantage-HF 2polymerase (Clontech Laboratories, Palo Alto Calif.) in 50 microlitervolume. The following reaction conditions were used:

a) 96° C.  3 minutes b) 96° C. 30 seconds denaturation c) 60° C. 30seconds, primer annealing d) 72° C.  3 minutes extension. Repeat steps(b)-(d) 45 times e) 72° C. 10 minutes final extension

The expected amplified product of approximately 1.9 kbp was detected byagarose gel electrophoresis. The fragment was isolated from the gel andligated to the vector pCR2.1 (Invitrogen, Carlsbad, Calif.) using M13Forward, M13 Reverse primers. The cloned insert was sequenced as PCRamplicons using the following gene-specific primers:

2864933-Seq-0CACAAGCCAGGACGGAACA (SEQ ID NO:35)

2864933-Seq-1 TGG AAC TAA TGC CTT CAA C (SEQ ID NO:36)

2864933-Seq-2 GAG TCCTGGAGAAACAGTGGA (SEQ ID NO:37)

2864933-Seq-3 ATGAGGCAGTGCCCTCCATC (SEQ ID NO:38)

2864933-Seq-4 CCATATTGTGGATGGATAA (SEQ ID NO:39)

2864933-Seq-5 GACACTCAATCCAAAGACC (SEQ ID NO:40)

2864933-Seq-6 CCATCACGCAGCAGGGCTA (SEQ ID NO:41)

The cloned cDNA (SEQ ID NO:29) was verified to have an open readingframe coding for the predicted mature extracellular domain of 2864933between residues 19 and 644 (SEQ ID NO:30)(FIG. 15). In FIG. 15, theBamHI and XhoI cloning sites, and the amino acids encoded by them (whichare therefore not part of the cloned sequence), are in bold font. Theconstruct is called pCR2.1-2864933.

Example 3 Expression of h2864933 in Human Embryonic Kidney 293 Cells.

Oligonucleotide primers pSec-V5-His Forward and pSec-V5-His Reverse weredesigned to amplify a fragment from the pcDNA3.1-VγHis (Invitrogen,Carlsbad, Calif.) expression vector. The PCR product was digested withXhoI and ApaI and ligated into the XhoI/ApaI digested pSecTag2 B vectorharboring an Ig kappa leader sequence (Invitrogen, Carlsbad Calif.). Thecorrect structure of the resulting vector, pSecV5His, was verified byDNA sequence analysis. The vector pSecV5His was digested with PmeI andNheI, and the PmeI-NheI fragment was ligated into the BamHI/Klenow andNheI treated vector pCEP4 (Invitrogen, Carlsbad, Calif.). The resultingvector was named pCEP4/Sec.

pSec-V5-His Forward

CTCGTCCTCGAGGGTAAGCCTATCCCTAAC (SEQ ID NO:42)

pSec-V5-His Reverse

CTCGTCGGGCCCCTGATCAGCGGGTTTAAAC (SEQ ID NO:43),

The 2 kb BamHI-XhoI fragment containing the h2864933 sequence wasisolated from pCR2.1-2864933 (Example 2) and subcloned into BamHI-XhoIdigested pCEP4/Sec to generate expression vector pCEP4/Sec-2864933. ThepCEP4/Sec-2864933 vector was transfected into 293 cells using theLipofectaminePlus reagent following the manufacturer's instructions(Gibco/BRL, Life Technologies, Inc., Rockville, Md.). The cell pelletand supernatant were harvested 72 hours after transfection and examinedfor h2864933 expression by Western blotting (reducing conditions) withan anti-V5 antibody. FIG. 16 shows that h2864933 secreted by 293 cellsis detected in two bands carrying the V5 epitope at about 70 kDa andabout 100-kDa. The 70 kDa band is presumed to represent theunglycosylated protein, and corresponds to the M_(r) expected for the626 residue clone with the addition of the V5 epitope. The programPROSITE predicts six N-glycosylation sites in the extracellular h286493domain. It is thought that the 100 kDa band originates from glycosylatedform(s) of the protein.

Example 4 Molecular Cloning of 3352358-1

The predicted open reading frame of clone 3352358-1 codes for a 653amino acid residue Type I transmembrane protein with the transmembranedomain predicted to lie between residues 522 and 551. The cDNA codingfor the extracellular segment of the predicted mature protein (i.e.,after cleavage of the signal peptide), has been cloned.

The secretory signal prediction method, GCG:SPSCAN—Eukaryote, predicts asignal peptidase cleavage site for 3352358-1 between residues 41 and 42.Accordingly, the following oligonucleotide primers were designed to PCRamplify the predicted mature extracellular domain of 3352358 fromresidue 42 to 486:

3352358CForward

CTCGTCGGATCCAACTGCCCCTCCGTCTGCTCGTGCAG (SEQ ID NO:44), and

3352358CReverse

CTCGTCGTCGACCGTGGTAGAGGTGGTATATGCCGGCTG (SEQ ID NO:45).

For cloning purposes, the forward primer includes an in frame BamHIrestriction site and the reverse primer contains an in frame SalIrestriction site.

Two separate PCR reactions were set up using 5 ng human testis and fetalbrain cDNA templates, respectively. The reaction mixtures contained 1microM of each of the 3352358CForward and 3352358CReverse primers, 5micromoles dNTP (Clontech Laboratories, Palo Alto Calif.) and 1microliter of 50× Advantage-HF 2 polymerase (Clontech Laboratories, PaloAlto Calif.) in 50 microliter volume. The following reaction conditionswere used:

a) 96° C.  3 minutes b) 96° C. 30 seconds denaturation c) 70° C. 30seconds, primer annealing. This temperature was gradually decreased by1° C./cycle d) 72° C.  3 minutes extension. Repeat steps (b)-(d) 10times e) 96° C. 30 seconds denaturation f) 60° C. 30 seconds annealingg) 72° C.  3 minutes extension Repeat steps (e)-(g) 25 times h) 72° C.10 minutes final extension

The expected amplified product of 1335 bp was detected by agarose gelelectrophoresis in both samples. The fragments were purified fromagarose gel and ligated to pCR2.1 vector (Invitrogen, Carlsbad, Calif.).Using M13 Forward and M13 Reverse vector primers and the following genespecific primers:

3352358 Seq-1 GTGCAGTAACCAGTTCAGCA (SEQ ID NO:46),

3352358 Seq-2 ACCTGTCCAAGCTGCGGGAG (SEQ ID NO:47),

3352358 Seq-3 TTGACGGGCTGGCTTCACTT (SEQ ID NO:48),

3352358 Seq-4 GACAGTGCTCAGCCACGCCT (SEQ ID NO:49),

the cloned insert was sequenced as PCR amplicons and verified as an openreading frame designated as 3352358-S153A. The nucleotide sequence (SEQID NO:31) obtained for this clone is shown in FIG. 17 Panel A. Thecloning sites are in underlined italic font. The sequence obtained forclone 3352358-S153A differs from the sequence expected for clone3352358-1 at six positions. These are indicated in FIG. 17A byunderlined bold font. The translated protein sequence (SEQ ID NO:32) forclone 3352358-S153A is given in FIG. 17 Panel B. Five of the sequencedifferences found at the nucleotide level are translated into amino aciddifferences, compared to the sequence expected for clone 3352358-1;these are likewise indicated in FIG. 17B by underlined bold font. (InFIG. 17B, the two amino acid residues encoded by the cloning sites ateach end are not shown. The first amino acid residue of FIG. 17B isencoded by nucleotides 7-9 of FIG. 17A.)

Example 5 Expression of h3352358 in Human Embryonic Kidney 293 Cells

The vector pCEP4/Sec was prepared as described in Example 3. A 1.3 kbfragment containing the h52358 sequence was isolated from pCR2.1-3352358(prepared in Example 4) by BamHI-SalI digestion and subcloned intoBamHI-XhoI digested pCEP4/Sec to generate the expression vectorpCEP4/Sec-3352358. The pCEP4/Sec-3352358 vector was transfected into 293cells using the LipofectaminePlus™ reagent following the manufacturer'sinstructions (Gibco/BRL). The cell pellet and supernatant were harvested72 hours after transfection and examined for h3352358 expression byWestern blotting (reducing conditions) with an anti-V5 antibody. FIG. 18shows that h3352358 secreted by 293 cells is detected in a band carryingthe V5 epitope at about 98 kDa. This band is presumed to represent theglycosylated form of the protein, since the program PROSITE predictseight N-glycosylation sites in the extracellular h3352358 domainpolypeptide.

Example 6 Expression of 2777610 in Tissues Determined by TaqMan™Analysis

The expression of 2777610 was evaluated by real time quantitative PCR intissues indicated in Table 3, below. The numbering in column 1 of Table3 corresponds to the lane order of the histograms in FIGS. 19A-C throughFIG. 23.

TABLE 3 Panel of cell types used in TaqMan ™ Analysis 1 Endothelialcells 2 Endothelial cells (treated) 3 Pancreas 4 Pancreatic ca. CAPAN 25 Adipose 6 Adrenal gland 7 Thyroid 8 Salivary gland 9 Pituitary gland10 Brain (fetal) 11 Brain (whole) 12 Brain (amygdala) 13 Brain(cerebellum) 14 Brain (hippocampus) 15 Brain (hypothalamus) 16 Brain(substantia nigra) 17 Brain (thalamus) 18 Spinal cord 19 CNS ca.(glio/astro) U87-MG 20 CNS ca. (glio/astro) U-118-MG 21 CNS ca. (astro)SW1783 22 CNS ca.* (neuro; met) SK-N-AS 23 CNS ca. (astro) SF-539 24 CNSca. (astro) SNB-75 25 CNS ca. (glio) SNB-19 26 CNS ca. (glio) U251 27CNS ca. (glio) SF-295 28 Heart 29 Skeletal muscle 30 Bone marrow 31Thymus 32 Spleen 33 Lymph node 34 Colon (ascending) 35 Stomach 36 Smallintestine 37 Colon ca. SW480 38 Colon ca.* (SW480 met) SW620 39 Colonca. HT29 40 Colon ca. HCT-116 41 Colon ca. CaCo-2 42 Colon ca. HCT-15 43Colon ca. HCC-2998 44 Gastric ca.* (liver met) NCI-N87 45 Bladder 46Trachea 47 Kidney 48 Kidney (fetal) 49 Renal ca. 786-0 50 Renal ca. A49851 Renal ca. RXF 393 52 Renal ca. ACHN 53 Renal ca. UO-31 54 Renal ca.TK-10 55 Liver 56 Liver (fetal) 57 Liver ca. (hepatoblast) HepG2 58 Lung59 Lung (fetal) 60 Lung ca. (small cell) LX-1 61 Lung ca. (small cell)NCI-H69 62 Lung ca. (s. cell var.) SHP-77 63 Lung ca. (large cell)NCI-H460 64 Lung ca. (non-sm. cell) A549 65 Lung ca. (non-s. cell)NCI-H23 66 Lung ca (non-s. cell) HOP-62 67 Lung ca. (non-s. cl) NCI-H52268 Lung ca. (squam.) SW 900 69 Lung ca. (squam.) NCI-H596 70 Mammarygland 71 Breast ca.* (pl. effusion) MCF-7 72 Breast ca.* (pl. ef)MDA-MB-231 73 Breast ca.* (pl. effusion) T47D 74 Breast ca. BT-549 75Breast ca. MDA-N 76 Ovary 77 Ovarian ca. OVCAR-3 78 Ovarian ca. OVCAR-479 Ovarian ca. OVCAR-5 80 Ovarian ca. OVCAR-8 81 Ovarian ca. IGROV-1 82Ovarian ca.* (ascites) SK-OV-3 83 Myometrium 84 Uterus 85 Placenta 86Prostate 87 Prostate ca.* (bone met) PC-3 88 Testis 89 MelanomaHs688(A).T 90 Melanoma* (met) Hs688(B).T 91 Melanoma UACC-62 92 MelanomaM14 93 Melanoma LOX IMVI 94 Melanoma* (met) SK-MEL-5 95 MelanomaSK-MEL-28 96 Melanoma UACC-257

In the PCR assay used, a fluorogenic probe, consisting of anoligonucleotide with both a reporter and a quencher dye attached, isannealed specifically to the target sequence between the forward andreverse primers. When the probe is cleaved by the 5′ nuclease activityof the DNA polymerase, the reporter dye is separated from the quencherdye and a sequence-specific signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the increase in fluorescence intensity is monitored duringthe PCR.

Probes and primers were designed according to Perkin Elmer Biosystem'sPrimer Express Software package (version I for Apple Computer'sMacintosh Power PC) using the sequence of 2777610 as input. Defaultsettings were used for reaction conditions and the following parameterswere set before selecting primers: primer concentration=250 nM, primermelting temperature (T_(m)) range=58°-60° C., primer optimal Tm=59° C.,maximum primer difference=2° C., probe does not have 5′ G, probe T_(m)must be 10° C. greater than primer T_(m), amplicon size 75 bp to 100 bp.The probes and primers selected (see below) were synthesized, doubleHPLC purified to remove uncoupled dye and evaluated by mass spectroscopyfor efficient coupling of reporter and quencher dyes to the 5′ and 3′ends of the probe, respectively.

PCR conditions: Sample RNA was provided from a broad range of normal andtumor tissues. The RNA from each tissue (poly A+ RNA, 2.8 pg) and fromthe cell lines (total RNA, 70 ng) was spotted in each well of a 96 wellPCR plate. PCR cocktails including the forward primer, reverse primerand a 2777610-specific probe (see below; and another set of primers anda probe for another gene to serve as a reference) were set up using 1×TaqMan™ PCR Master Mix for the PE Biosystems 7700, with 5 mM MgCl2,dNTPs (dA, dG, dC, dU at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq Gold™ (PEBiosystems), and 0.4 U/μl RNase inhibitor, and 0.25 U/μl reversetranscriptase. Reverse transcription was performed at 48° C. for 30minutes followed by amplification/PCR cycles as follows: 95° C. 10 min,then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute.

TaqMan Probes and Primers Used in Analysis:

Ag 111 (F): 5′-CCTTTCAAAATCCTCTCTGACTCAC-3′ (SEQ ID NO:50)

Ag 111 (R): 5′-TCACCGAAGAAAAACGACACAC-3′ (SEQ ID NO:51)

Ag 111 (P): TET-5′-CCTGGCACCCTGGCAGCTCAGA-3′-TAMRA (SEQ ID NO:52)

Example 7 Expression of 2864933 in Tissues Determined by TaqMan™Analysis

TaqMan™ analysis of the expression of 2864933 was carried out asdescribed in Example 6. Reverse transcription was performed at 48° C.for 30 minutes followed by amplification/PCR cycles as follows: 95° C.10 min, then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute.Primer-probe sets are as described below. The results are shown in FIG.19, Panels A, B, and C, respectively. Cell types for each panel in FIG.19 are as provided in Table 3, above.

Two sets of primers and a probe targeted regions of the nucleic acidsthat are common to 2864933-1 and 2864933-2. Primer-probe set 88 includesAg 88 (SEQ ID NOs:53-55) and primer-probe set 291 includes Ag 291(SEQ IDNOs:56-58).

Ag 88 (F): 5′-CATCTTCAACAGGCCATGGTT-3′ (SEQ ID NO:53)

Ag 88 (R): 5′-AGCAGCTGTGTCCACTGCAA-3′ (SEQ ID NO:54)

Ag 88 (P): TET-5′-TGAGAACAATGGTCAGATACCGCCTTACCAA-3′-TAMRA (SEQ IDNO:55)

Ag 291 (F): 5′-CGCAGTCATTTACCGGAGTCTT-3′ (SEQ ID NO:56)

Ag 291 (R): 5′-TTCTTTCAACCATTTTGAATCGTG-3′ (SEQ ID NO:57)

Ag 291 (P): TET-5′-AGCCCTACCCTGCGGACCGTCA-3′-TAMRA (SEQ ID NO:58)

A third set of primers and a probe targeted the segment that isspecifically present only in the longer splice variant, 2864933-1.Primer-probe set 341 includes Ag 341 (SEQ ID NOs:59-61).

Ag 341 (F): 5′-TCCTTTGTGGCACTGAATGG-3′ (SEQ ID NO:59)

Ag 341 (R): 5′-CCCTCTTGAGCCGTCGAA-3′ (SEQ ID NO:60)

Ag 341 (P): FAM-5′-TCCCTCTTGCCCAGCACAACCAC-3′-TAMRA (SEQ ID NO:61)

Example 8 Expression of 3352358 in Tissues Determined by TaqMan™Analysis

TaqMan™ analysis of the expression of 3352358 was carried out asdescribed in Example 6, using the tissue panel as described in Table 3.Reverse transcription was performed at 48° C. for 30 minutes followed byamplification/PCR cycles as follows: 95° C. 10 min, then 40 cycles of95° C. for 15 seconds, 60° C. for one (1) minute.

TaqMan™ probes and primers used in Analysis of 3352358 are shown below(primer and probe annealing positions to sequence input shown in colorand underline respectively). Primer-probe set 42 includes Ag 42 (SEQ IDNOs:62-64). The results are shown in FIG. 20.

Ag 42 (F): 5′-CGCGAAAGTACAAGCCTGTTC-3′ (SEQ ID NO:62)

Ag 42 (R): 5′-GAATGAGCACCGTGGTAGAGG-3′ (SEQ ID NO:63)

Ag 42 (P): TET-5′-CGTCCACTGGTTACCAGCCGGCATATA-3′-TAMRA (SEQ ID NO:64)

Example 9 Expression of 3911675 in Tissues Determined by TaqMan™Analysis

TaqMan™ analysis of the expression of 3911675 was carried out asdescribed in Example 6, using the tissue panel as described in Table 3.Reverse transcription was performed at 48° C. for 30 minutes followed byamplification/PCR cycles as follows: 95° C. 10 min, then 40 cycles of95° C. for 15 seconds, 60° C. for 1 minute. TaqMan Probes and PrimersUsed in the expression Analysis of 3911675 are the primer-probe set 115,which includes Ag 15 (SEQ ID NOs:65-67). The results are shown in FIG.21.

Ag 115 (F): 5′-TGGACTCATCCCACTTGCTCT-3′ (SEQ ID NO:65)

Ag 115 (R): 5′-CCTGCGCAAAAAGTTGTGAA-3′ (SEQ ID NO:66)

Ag 115 (P): TET-5′-CAGCTGAATCCTGACATCATATCCACACTGTGT-3′-TAMRA (SEQ IDNO:67)

Example 10 Expression of 4035508 in Tissues Determined by TaqMan™Analysis

TaqMan™ analysis of the expression of 4035508 was carried out asdescribed in Example 6, using the tissue panel as described in Table 3.Reverse transcription was performed at 48° C. for 30 minutes followed byamplification/PCR cycles as follows: 95° C. 10 min, then 40 cycles of95° C. for 15 seconds, 60° C. for 1 minute. TaqMan probes and primersused in the expression analysis for clone 4035508 include theprimer-probe set 118, termed Ag 118 (SEQ ID NOs:68-70). The results areshown in FIG. 22.

Ag 118 (F): 5′-TCTCTGTCTGCAGTACCTGGCAT-3′ (SEQ ID NO:68)

Ag 118 (R): 5′-GGCAGTGGGTATGGGATGTG-3′ (SEQ ID NO:69)

Ag 118 (P): FAM-5′-ACTTTCCTCCTGATGCCCCGGG-3′-TAMRA (SEQ ID NO:70)

Example 11 Expression of 4339264 in Tissues Determined by TaqMan™Analysis

TaqMan™ analysis of the expression of 4339264 was carried out asdescribed in Example 6, using the tissue panel as described in Table 3.Reverse transcription was performed at 48° C. for 30 minutes followed byamplification/PCR cycles as follows: 95° C. 10 min, then 40 cycles of95° C. for 15 seconds, 60° C. for 1 minute. TaqMan probes and primersused in the expression analysis for clone 4339264 include theprimer-probe set 120, termed Ag 120 (SEQ ID NOs:71-73). The results areshown in FIG. 23.

Ag 120 (F): 5′-AAAGGCGGAGGAAAGAAGTACTC-3′ (SEQ ID NO:71)

Ag 120 (R): 5′-GCTCCCGTTCCCTCTCCA-3′ (SEQ ID NO: 72)

Ag 120 (P): FAM-5′-CCTCTTTGTTCTTCTTGCCCGAGTTTTCTTT-3′-TAMRA (SEQ ID NO:73)

Equivalents

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique nucleotides,polypeptides, and methods of use thereof for the SECX genes have beendescribed. Although particular embodiments have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims which follow. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention as defined by the claims. Forinstance, the choice of which SECX nucleotide or polypeptide or methodof use thereof is believed to be a matter of routine for a person ofordinary skill in the art with knowledge of the embodiments describedherein.

1. An isolated nucleic acid comprising: (a) a nucleic acid sequenceencoding a polypeptide of SEQ ID NO: 10; or (b) a nucleic acid encodinga polypeptide wherein the polypeptide has conservative amino acidsubstitutions to the polypeptide of SEQ ID NO:10 and wherein thepolypeptide has neural development activity.
 2. The nucleic acid ofclaim 1, wherein said nucleic acid is DNA or RNA.
 3. The nucleic acid ofclaim 1, wherein said nucleic acid comprises an open reading frame thatencodes a polypeptide of SEQ ID NO:
 10. 4. The nucleic acid of claim 1,wherein said nucleic acid comprises a nucleic acid sequence which is SEQID NO: 9 or its complement.
 5. A vector comprising the nucleic acid ofclaim
 1. 6. A cell comprising the vector of claim
 5. 7. The cell ofclaim 6 wherein said cell is a prokaryotic or eukaryotic cell omprisingthe nucleic acid sequence which is SEQ ID NO: 9, or its complement.
 8. Apharmaceutical composition comprising the nucleic acid of claim 1 and apharmaceutically acceptable carrier.
 9. A process for producing apolypeptide encoded by the nucleic acid of claim 1, said processcomprising: (a) providing the cell of claim 6; (b) culturing said cellunder conditions sufficient to express said polypeptide; and (c)recovering said polypeptide, thereby producing said polypeptide.
 10. Theprocess of claim 9 wherein said cell is a prokaryotic or eukaryoticcell.
 11. A process for identifying a compound that binds the nucleicacid of claim 1, the process comprising: (a) contacting said nucleicacid with a compound; and (b) determining whether said compound bindssaid nucleic acid sequence.
 12. A compound identified by the process ofclaim 11.